23413, a novel human ubiquitin protease

ABSTRACT

The present invention relates to a newly identified human ubiquitin protease belonging to the family of mammalian deubiquitinating enzymes. The invention also relates to polynucleotides encoding the ubiquitin protease. The invention further relates to methods using the ubiquitin protease polypeptides and polynucleotides as a target for diagnosis and treatment in ubiquitin-mediated or -related disorders. The invention further relates to drug-screening methods using the ubiquitin protease polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the ubiquitin protease polypeptides and polynucleotides. The invention further relates to procedures for producing the ubiquitin protease polypeptides and polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Application Ser. No.09/406,045, filed Sep. 27, 1999, which is hereby incorporated in itsentirety by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to a newly identified humanubiquitin protease belonging to the family of mammalian deubiquitinatingenzymes. The invention also relates to polynucleotides encoding theubiquitin protease. The invention further relates to methods using theubiquitin protease polypeptides and polynucleotides as a target fordiagnosis and treatment in ubiquitin-mediated or -related disorders. Theinvention further relates to drug-screening methods using the ubiquitinprotease polypeptides and polynucleotides to identify agonists andantagonists for diagnosis and treatment. The invention furtherencompasses agonists and antagonists based on the ubiquitin proteasepolypeptides and polynucleotides. The invention further relates toprocedures for producing the ubiquitin protease polypeptides andpolynucleotides.

BACKGROUND OF THE INVENTION

[0003] The Ubiquitin System

[0004] Several biological processes are controlled by the ubiquitinationof cellular protein. Cellular processes that are affected by ubiquitinmodification include the regulation of gene expression, regulation ofthe cell cycle and cell division, cellular housekeeping, cell-specificmetabolic pathways, disposal of mutated or post-translationally damagedproteins, the cellular stress response, modification of cell surfacereceptors, DNA repair, import of proteins into mitochondria, uptake ofprecursors into neurons, biogenesis of mitochondria, ribosomes, andperoxisomes, apoptosis, and growth factor-mediated signal transduction.

[0005] For some protein substrates ubiquitination leads to proteindegradation by the 26S proteasomal complex. A wide variety of proteinsubstrates is degraded by the 26S proteasomal complex followingubiquitination of the substrate. Degradation of a protein by theubiquitin system involves two steps. The first involves the covalentattachment of multiple ubiquitin molecules to the substrate protein. Thesecond involves degradation of the ubiquitinated protein by the 26Sproteasome. In some cases, degradation of the ubiquitinated protein canoccur by means of the lysosomal pathway.

[0006] The 26S proteasome comprises a 20S core catalytic complex whichis flanked by two 19S regulatory complexes. The 26S complex recognizesubiquitinated proteins. Substrate recognition by the 26S proteasome,however, may be mediated by the interaction of specific subunits of the19S complex with the ubiquitin chain. The ubiquitinated protein isdegraded by specific and energy-dependent proteases into free aminoacids and free and reutilizable ubiquitin.

[0007] The 19S regulatory complex consists of many subunits that can beclassified into ATPases and non-ATPases. This complex is thought to actin recognition, unfolding, and translocation of the substrates into the20S proteasome for proteolysis. The regulatory complex containsisopeptidases capable of deubiquitinating substrates (Spataro et al.(1998) British Journal of Cancer 77:448-455).

[0008] The ubiquitin proteasome pathway functions to degrade abnormalproteins, short-lived normal proteins, long-lived normal proteins, andproteins of the endoplasmic recticulum. Important regulatory proteinsrapidly inactivated by proteolysis include c-JUN, c-FOS, and p53 (Leckeret al. (1999) Journal of Nutrition 129:227S-237S). Conditions thatstimulate protein degradation by the ubiquitin proteasome pathwayinclude eating disorders, renal tubular defects, diabetes, uremia,neuromuscular disease, immobilization, burn injuries, sepsis, cancer,cachexia, hyperadrenocortisolism and hyperthyroidism.

[0009] Cellular proteins degraded by the ubiquitin system include cellcycle regulators, including mitotic cyclins, G1 cyclins, CDK inhibitors,anaphase inhibitors, transcription factors, tumor suppressors, andoncoproteins such as NF-ηB and IηBα, p53, JUN, β-catenin, E2F-1, andmembrane proteins such as Ste2p, GH receptor, T-cell receptor,platelet-derived growth factor, lymphocyte homing receptor, MET tyrosinekinase receptor, hepatocyte growth factor-scatter factor, connexin 43,the high affinity IgE receptor, the prolactin receptor, and the EGFreceptor (Hershko et al. (1998) Annual Review of Biochemistry67:425-479).

[0010] Ubiquitination does not only result in proteolytic degradation.For some protein substrates, ubiquitination is a reversiblepost-translational modification that can regulate cellular targeting andenzymatic activity. This includes targeting to the vacuole, activationof enzyme activity, such as Ikβ kinase activation, and activation ofcytokine receptor-mediated signal transduction (D'Andrea et al. (1998)Critical Reviews In Biochemistry and Molecular Biology 33:337-352). TheT-cell receptor undergoes ubiquitination in response to receptorengagement. Platelet derived growth factor undergoes multipleubiquitination following ligand binding. Soluble steel factor has beenshown to stimulate rapid polyubiquitination of the c-KIT receptor.

[0011] It has been shown that protein degradation accounts forregulation of proteins such as cyclins, cyclin-dependent kinaseinhibitors, p53, c-JUN and c-FOS (Spitaro et al. above). The ubiquitinsystem has also shown to be involved in antigen presentation. The 26Sproteasome is responsible for processing MHC-restricted class I antigens(Spitaro et al. above).

[0012] The ubiquitin system has been implicated in various diseases. Onegroup includes pathology that results from loss of function, a mutationin an enzyme or substrate that leads to stabilization of the protein andconsequent build up of a protein to abnormally high levels. The secondinvolves pathologies that result from a gain of function that producesincreased protein degradation.

[0013] The ubiquitin system has been implicated in various malignancies.In cervical carcinoma, low levels of p53 have been found. This proteinis targeted for degradation by HPV E6-associated protein. Removal of thesuppressor by this oncoprotein may be a mechanism utilized by the virusto transform cells. Other results have shown that c-JUN, but not thetransforming counterpart, v-JUN, is ubiquitinated and subsequentlydegraded. Other studies show that low levels of p27, a cell divisionkinase inhibitor whose degradation is necessary for proper cell cycleprogression, is correlated with colorectal, and breast carcinomas. Thelow level of this enzyme is due to activation of the ubiquitin system.

[0014] Human genetic diseases involving aberrant proteolysis have beenreviewed (Kato (1999) Human Mutation 13:87-98). Cystic fibrosis has beencorrelated with the ubiquitin system. The cystic fibrosis transmembraneregulator in cystic fibrosis patients is almost completely degraded bythe ubiquitin system so that an abnormally low amount of the wild typeprotein is found on the cell surface. In Angelman's syndrome, one of theenzymes involved in ubiquitination (E3) is affected. In Liddle syndrome,the E3 enzyme is also affected.

[0015] The ubiquitin system can also affect the immune and inflammatoryresponse. The persistence of EBNA-1 contributes to some virus relatedpathologies. A sequence on this protein was found to inhibit degradationby the ubiquitin system. This inhibited processing and subsequentpresentation of viral epitopes by MHC protein.

[0016] The ubiquitin system has also been implicated inneurodegenerative diseases. Ubiquitin immunohistochemistry has shownenrichment of ubiquitin conjugates in senile plaques, lysosomes,endosomes, and a variety of inclusion bodies and degenerative fibers inmany neurodegenerative diseases, such as Alzheimer's, Parkinson's andLewy body diseases, amyotrophic lateral sclerosis, and Creutzfeld-Jakobdisease. Further, in Huntington disease and spinocerebellar ataxias, theproteins encoded by the affected genes aggregate in ubiquitin- andproteasome-positive intranuclear inclusion bodies.

[0017] The ubiquitin system has been associated with muscle wasting(Mitch et al. (1999) American Journal of Physiology 276:C1132-C1138 andLecker et al. above) and muscle-wasting diseases and in suchpathological states as fasting, starvation, sepsis, and denervation, allof which result from accelerated ubiquitin-mediated proteolysis (seeCiechanover, EMBO Journal 17:7151-7160 (1998)).

[0018] The ubiquitin system is also involved in development. Theinvolvement in human brain development is indicated by the fact that amutation in an E3 enzyme is implicated as the cause of Angelman'ssyndrome, a disorder characterized by mental retardation, seizures, andabnormal gait (Hershko et al. above).

[0019] The ubiquitin system is also associated with apoptosis.Ubiquitin-proteasome-mediated proteolysis is reported to play animportant role in apoptosis of nerve growth factor-deprived neurons(Sadoul et al. (1996) EMBO Journal 15:3845-3852). One of the first genesshown to be involved in programmed cell death is the polyubiquitin genethat is regulated during metamorphosis of Manduca sexta.Radiation-induced apoptosis in human lymphocytes has been shown to beaccompanied by increased ubiquitin mRNA and ubiquitinylated nuclearproteins. Further, drugs that interfere with proteasome function, suchas lactacystin, prevent radiation-induced cell death of thymocytes(Hershko et al. above).

[0020] Deubiquitinating Enzymes

[0021] Deubiquitinating enzymes are cysteine proteases that specificallycleave ubiquitin conjugates at the ubiquitin carboxy terminus. Theseenzymes are responsible for processing linear polyubiquitin chains togenerate free ubiquitin from precursor fusion proteins. They also affectpools of free ubiquitin by recycling branched chain ubiquitin. Theseenzymes also remove ubiquitin from ubiquitin- andpolyubiquitin-conjugated target protein, thereby regulating localizationor activity of the target. Further, these enzymes can remove ubiquitinfrom a ubiquitinated tagged protein and thereby rescue the protein fromdegradation by the 26S proteasome. The end result of each of theseactivities, is to affect the level of free intracellular ubiquitin(D'Andrea et al., above) and the level of specific proteins.

[0022] Ubiquitin is synthesized in a variety of functionally-distinctforms. One of these is a linear head-to-tail polyubiquitin precursor.Release of the free molecules involves specific enzymatic cleavagebetween the fused residues. The last ubiquitin moiety in many of theseprecursors is encoded with an extra C-terminal residue that must beremoved to expose the active C-terminal Gly. In general, the recyclingenzymes are thiol proteases that recognize the C-terminal domain/residueof ubiquitin. These are divided into two classes. The first isdesignated ubiquitin C-terminal hydrolase (UCH) and the second isdesignated ubiquitin-specific protease (UBP; isopeptidases)(Ciechanover, above). These enzymes have been reviewed in detail inD'Andrea, above.

[0023] UBPs contain six conserved regions. One surrounds the conservedcysteine, one surrounds the aspartic acid, one surrounds the histidine,and three additional regions of unknown function have been identified.These six domains provide a molecular signature for the UBP family.Short sequences surrounding the cysteine residue and histidine residueare highly conserved among all UBPs. Sequence comparison of several UBPfamily members reveals that there are various subfamilies. Onesubfamily, designated DUB, contains enzymes that are transcriptionallyinduced in response to cytokines. The UBP family contains enzymes whosemembers have multiple ubiquitin binding sites. Identified members ofthis family include DUB1, isoT, UBP3, Doa4, Tre2, and FAF (D'Andrea etal. above).

[0024] The UCH family is distinct from the UBP family. These enzymes arecysteine proteases but do not contain the six homology domainscharacteristic of the UBP family. Further, there is only one bindingsite for ubiquitin. With respect to substrate specificity, the UCHfamily preferentially cleaves ubiquitin from small molecules, such aspeptides and amino acids. Further, the two families share littlesequence homology with each other, although the UCH signature can befound in some UBPs.

[0025] The deubiquitinating enzymes can promote either degradation orstabilization of a given substrate. One of the best characterizeddeubiquitinating enzymes is the yeast UBP14p enzyme which has a humanhomolog designated isopeptidase-T. Isopeptidase-T hydrolyzes freepolyubiquitin chains and stimulates degradation of polyubiquitinatedprotein substrates by the 26S proteasome. In vitro data suggest that thecellular role of isopeptidase-T is to dissemble unanchored polyubiquitinchains. The isopeptidase-T then sequentially degrades thesepolyubiquitin chains into ubiquitin monomers.

[0026] The yeast Doa4 promotes ubiquitin-mediated proteolysis ofcellular substrates. The primary function appears to be the hydrolysisof isopeptide-linked ubiquitin chains from peptides that are theby-products of proteasome degradation. The function appears to be theclipping of polymeric ubiquitin from peptide degradation products. Insummary, with respect to a degradation function, isopeptidases canproduce free ubiquitin monomers from straight chain polyubiquitin,branched chain polyubiquitin, ubiquitin or polyubiquitin attached tosubstrate proteins, and ubiquitin or polyubiquitin attached to substrateremnants, such as peptides or amino acids.

[0027] Deubiquitinating enzymes that promote stabilization of substratesinclude the FAF protein. Results show that the FAF proteindeubiquitinates and rescues a ubiquitin-conjugated target, preventingits degradation by the proteasome. Another deubiquitinating enzyme,designated PA700 isospeptidase, also prevents proteasome degradation.This enzyme has been isolated from the 19S regulatory complex. Thisenzyme appears to remove one ubiquitin at a time starting from thedistal end of a polyubiquitin chain.

[0028] The enzymes have been associated with growth control. Themammalian oncoprotein Tre-2 is a member of the UBP superfamily. Thetransforming isoform of the Tre-2 oncoprotein is a truncated UPB lackingthe histidine domain and lacking deubiquitinating activity. The fulllength Tre-2 protein has deubiquitinating activity but no transformingactivity. Accordingly, it has been suggested that this protein acts as agrowth suppressor within the cell.

[0029] Another UBP that regulates cellular function is designated DUB.DUB-1 was originally shown to be induced by interleukin-3 stimulation.It has been postulated that the DUB protein family is generallyresponsive to cytokines. It has also been shown that another familymember, DUB-2, is induced by interleukin-2. Zhu et al. (1997) Journal ofBiological Chemistry 272:51-57.

[0030] The enzymes may deubiquitinate cell surface growth factorreceptors thereby prolonging receptor half life and amplifying growthsignals. They may also deubiquitinate proteins involved in signaltransduction and deubiquitinate cell cycle regulators such as cyclins orcyclin-CDK inhibitors. See D'Andrea above.

[0031] UBPs have also been linked to the chromatin regulatory process,transcriptional silencing. UBP-3 has been reported to complex withSIR-4, a trans-acting factor that is required for establishment andmaintenance of silencing. Accordingly, UBP-3 may act as an inhibitor ofsilencing by either stabilizing an inhibitor or by removing a positiveregulator.

[0032] The murine UNP protooncogene has been shown to encode a nuclearubiquitin protease whose overexpression leads to oncogenictransformation in NIH3T3 cells. A cDNA was cloned corresponding to thehuman homolog of this gene. It was shown to map to a region frequentlyrearranged in human tumor cells. Further, it was shown that levels ofthis gene are elevated in small cell tumors and adenocarcinomas of thelung, suggesting a causative role of the gene in the neoplastic process(Gray et al. (1995) Oncogene 10:2179-2183).

[0033] A novel ubiquitin-specific protease, designated UBP-43, wascloned from a leukemia fusion protein in AML1-ETO Knockin mice. Thisprotease was shown to function in hematopoitic cell differentiation. Theoverexpression of this gene was shown to block cytokine-induced terminaldifferentiation of monocytic cells (Liu et al. (1999) Molecular andCellular Biology 19:3029-3038).

[0034] In summary, deubiquitinating enzymes are potentially powerfultargets for modulating ubiquitination. Modulation of ubiquitination canincrease or decrease the proteolysis of specific proteins, particularlykey proteins in cellular processes, can increase or decrease levels ofgeneral proteolysis, thus affecting the basic metabolic state, and mayincrease or decrease the pool of free ubiquitin monomers available forubiquitination.

[0035] Accordingly, ubiquitin proteases are a major target for drugaction and development. Thus, it is valuable to the field ofpharmaceutical development to identify and characterize previouslyunknown ubiquitin proteases. The present invention advances the state ofthe art by providing a previously unidentified human deubiquitinatingenzyme.

SUMMARY OF THE INVENTION

[0036] It is an object of the invention to identify novel ubiquitinproteases.

[0037] It is a further object of the invention to provide novelubiquitin protease polypeptides that are useful as reagents or targetsin assays applicable to treatment and diagnosis of ubiquitin-mediated or-related disorders, especially disorders mediated by or related todeubiquitinating enzymes.

[0038] It is a further object of the invention to providepolynucleotides corresponding to the novel ubiquitin proteasepolypeptides that are useful as targets and reagents in assaysapplicable to treatment and diagnosis of ubiquitin or ubiquitinprotease-mediated or -related disorders and useful for producing novelubiquitin protease polypeptides by recombinant methods.

[0039] A specific object of the invention is to identify compounds thatact as agonists and antagonists and modulate the expression of the novelubiquitin protease.

[0040] A further specific object of the invention is to providecompounds that modulate expression of the ubiquitin protease fortreatment and diagnosis of ubiquitin and ubiquitin protease-relateddisorders.

[0041] The invention is thus based on the identification of a novelhuman ubiquitin protease. The amino acid sequence is shown in SEQ IDNO:1. The nucleotide sequence is shown in SEQ ID NO:2.

[0042] The invention provides isolated ubiquitin protease polypeptides,including a polypeptide having the amino acid sequence shown in SEQ IDNO:1 or the amino acid sequence encoded by the cDNA deposited with thePatent Depository of the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va., on Apr. 6, 2000, and assignedPatent Deposit No. PTA-1652 (“the deposited cDNA”). This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112. The deposited sequences, as well as thepolypeptides encoded by the sequences, are incorporated herein byreference and controls in the event of any conflict, such as asequencing error, with description in this application.

[0043] The invention also provides isolated ubiquitin protease nucleicacid molecules having the sequence shown in SEQ ID NO:2 or in thedeposited cDNA.

[0044] The invention also provides variant polypeptides having an aminoacid sequence that is substantially homologous to the amino acidsequence shown in SEQ ID NO:1 or encoded by the deposited cDNA.

[0045] The invention also provides variant nucleic acid sequences thatare substantially homologous to the nucleotide sequence shown in SEQ IDNO:2 or in the deposited cDNA.

[0046] The invention also provides fragments of the polypeptide shown inSEQ ID NO:1 and nucleotide sequence shown in SEQ ID NO:2, as well assubstantially homologous fragments of the polypeptide or nucleic acid.

[0047] The invention further provides nucleic acid constructs comprisingthe nucleic acid molecules described herein. In a preferred embodiment,the nucleic acid molecules of the invention are operatively linked to aregulatory sequence.

[0048] The invention also provides vectors and host cells for expressingthe ubiquitin protease nucleic acid molecules and polypeptides, andparticularly recombinant vectors and host cells.

[0049] The invention also provides methods of making the vectors andhost cells and methods for using them to produce the ubiquitin proteasenucleic acid molecules and polypeptides.

[0050] The invention also provides antibodies or antigen-bindingfragments thereof that selectively bind the ubiquitin proteasepolypeptides and fragments.

[0051] The invention also provides methods of screening for compoundsthat modulate expression or activity of the ubiquitin proteasepolypeptides or nucleic acid (RNA or DNA).

[0052] The invention also provides a process for modulating ubiquitinprotease polypeptide or nucleic acid expression or activity, especiallyusing the screened compounds. Modulation may be used to treat conditionsrelated to aberrant activity or expression of the ubiquitin proteasepolypeptides or nucleic acids or of the ubiquitin system.

[0053] The invention also provides assays for determining the activityof or the presence or absence of the ubiquitin protease polypeptides ornucleic acid molecules in a biological sample, including for diseasediagnosis.

[0054] The invention also provides assays for determining the presenceof a mutation in the polypeptides or nucleic acid molecules, includingfor disease diagnosis.

[0055] In still a further embodiment, the invention provides a computerreadable means containing the nucleotide and/or amino acid sequences ofthe nucleic acids and polypeptides of the invention, respectively.

DESCRIPTION OF THE DRAWINGS

[0056]FIG. 1 shows the nucleotide sequence (SEQ ID NO:2) and the deducedamino acid sequence (SEQ ID NO:1) of the novel ubiquitin protease. Theunderlined amino acids designate the conserved cysteine region andconserved histidine region. These regions are conserved among members ofthe UBP protein family.

[0057]FIG. 2 shows a comparison of the ubiquitin protease against theProDom database of protein patterns (SEQ ID NOS:3, 4, 5, 6, 7, 8, 9, 10,11 and 12), specifically showing some homology to the UCH family.

[0058]FIG. 3 shows an analysis of the ubiquitin protease amino acidsequence: αβturn and coil regions; hydrophilicity; amphipathic regions;flexible regions; antigenic index; and surface probability plot.

[0059]FIG. 4 shows a hydrophobicity plot of the ubiquitin protease ofSEQ ID NO:1.

[0060]FIG. 5 shows an analysis of the ubiquitin protease open readingframe for amino acids corresponding to specific functional sites of SEQID NO:1. Glycosylation sites are found from about amino acid 188 toabout amino acid 191 and from about amino acid 335 to about amino acid338, with the actual modified residue being the first amino acid. CyclicAMP and cyclic GMP-dependent protein kinase phosphorylation sites arefound from about amino acid 84 to about amino acid 87 and from aboutamino acid 288 to about amino acid 291, with the actual modified residuebeing the last amino acid. Protein kinase C phosphorylation sites arefound from about amino acid 169 to about amino acid 171, from aboutamino acid 185 to about amino acid 187, from about amino acid 223 toabout amino acid 225, from about amino acid 260 to about amino acid 262,and from about amino acid 266 to about amino acid 268, with the actualmodified residue being the first amino acid. Casein kinase IIphophorylation sites are found from about amino acid 22 to about aminoacid 25, from about amino 197 to about amino acid 200, from about aminoacid 208 to about amino acid 211, and from about amino acid 343 to aboutamino acid 346, with the actual modified residue being the first aminoacid. A tyrosine kinase phosphorylation site is found from about aminoacid 119 to about amino acid 125, with the actual modified residue beingthe last amino acid. N-myristoylation sites are found from about aminoacid 61 to about amino acid 66, and from about amino acid 312 to aboutamino acid 317, with the actual modified residue being the first aminoacid. An amidation site is found from about amino acid 233 to aboutamino acid 236. In addition, amino acids corresponding to the UCHsignature are found at amino acids 302-319.

[0061]FIG. 6 shows expression of the protease in normal breast, lung,liver, and colon, and shows enhanced expression in malignant breast,lung, liver, and colon metastases. The liver metastases are derived frommalignant colonic tissue.

[0062]FIG. 7 shows expression of the protease in cDNA libraries fromvarious tissues and cell types in culture. These data were derived fromRT-PCR of various cDNA libraries and hence, are not relativelyquantitative.

[0063]FIG. 8 shows expression of the protease in various normal humantissues. Expression was obtained by Taqman analysis and hence arerelatively quantitative.

DETAILED DESCRIPTION OF THE INVENTION

[0064] Polypeptides

[0065] The invention is based on the identification of a novel humanubiquitin protease. Specifically, an expressed sequence tag (EST) wasselected based on homology to ubiquitin protease sequences. This EST wasused to design primers based on sequences that it contains and used toidentify a cDNA from an endothelial cell cDNA library. Positive cloneswere sequenced and the overlapping fragments were assembled. Analysis ofthe assembled sequence revealed that the cloned cDNA molecule encodes aubiquitin protease containing the conserved HIS and CYS boxes of the UBPfamily of deubiquitinating enzymes.

[0066] The invention thus relates to a novel ubiquitin protease havingthe deduced amino acid sequence shown in FIG. 1 (SEQ ID NO:1) or havingthe amino acid sequence encoded by the deposited cDNA, ATCC PatentDeposit No. PTA-1652. “Ubiquitin protease polypeptide” or “ubiquitinprotease protein” refers to the polypeptide in SEQ ID NO:1 or encoded bythe deposited cDNA. The term “ubiquitin protease protein” or “ubiquitinprotease polypeptide”, however, further includes the numerous variantsdescribed herein, as well as fragments derived from the full-lengthubiquitin proteases and variants.

[0067] Tissues and/or cells in which the ubiquitin protease nucleic acidis found include, but are not limited to, those shown in FIGS. 6, 7, and8. Tissues in which the gene is highly expressed include breast, testes,liver, and fetal liver. The gene is also significantly expressed inthymus, brain, skeletal muscle, prostate, thyroid, fetal kidney, fetalheart, and ovary. The ubiquitin protease is particularly expressed intissues involved in breast and lung cancer. The gene is alsoparticularly expressed in liver metastases. These liver metastases arederived from malignant colonic tissue. Expression has been confirmed byNorthern blot analysis.

[0068] The present invention thus provides an isolated or purifiedubiquitin protease polypeptide and variants and fragments thereof.

[0069] Based on a BLAST search, highest homology was shown to murineUBP43 (Liu, above).

[0070] As used herein, a polypeptide is said to be “isolated” or“purified” when it is substantially free of cellular material when it isisolated from recombinant and non-recombinant cells, or free of chemicalprecursors or other chemicals when it is chemically synthesized. Apolypeptide, however, can be joined to another polypeptide with which itis not normally associated in a cell and still be considered “isolated”or “purified.”

[0071] The ubiquitin protease polypeptides can be purified tohomogeneity. It is understood, however, that preparations in which thepolypeptide is not purified to homogeneity are useful and considered tocontain an isolated form of the polypeptide. The critical feature isthat the preparation allows for the desired function of the polypeptide,even in the presence of considerable amounts of other components. Thus,the invention encompasses various degrees of purity.

[0072] In one embodiment, the language “substantially free of cellularmaterial” includes preparations of the ubiquitin protease having lessthan about 30% (by dry weight) other proteins (i.e., contaminatingprotein), less than about 20% other proteins, less than about 10% otherproteins, or less than about 5% other proteins. When the polypeptide isrecombinantly produced, it can also be substantially free of culturemedium, i.e., culture medium represents less than about 20%, less thanabout 10%, or less than about 5% of the volume of the proteinpreparation.

[0073] A ubiquitin protease polypeptide is also considered to beisolated when it is part of a membrane preparation or is purified andthen reconstituted with membrane vesicles or liposomes.

[0074] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the ubiquitin protease polypeptidein which it is separated from chemical precursors or other chemicalsthat are involved in its synthesis. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of the polypeptide having less than about 30% (by dryweight) chemical precursors or other chemicals, less than about 20%chemical precursors or other chemicals, less than about 10% chemicalprecursors or other chemicals, or less than about 5% chemical precursorsor other chemicals.

[0075] In one embodiment, the ubiquitin protease polypeptide comprisesthe amino acid sequence shown in SEQ ID NO:1. However, the inventionalso encompasses sequence variants. Variants include a substantiallyhomologous protein encoded by the same genetic locus in an organism,i.e., an allelic variant.

[0076] Variants also encompass proteins derived from other genetic lociin an organism, but having substantial homology to the ubiquitinprotease of SEQ ID NO:1. Variants also include proteins substantiallyhomologous to the ubiquitin protease but derived from another organism,i.e., an ortholog. Variants also include proteins that are substantiallyhomologous to the ubiquitin protease that are produced by chemicalsynthesis. Variants also include proteins that are substantiallyhomologous to the ubiquitin protease that are produced by recombinantmethods. It is understood, however, that variants exclude any amino acidsequences disclosed prior to the invention.

[0077] As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences are at leastabout 70-75%, typically at least about 80-85%, and most typically atleast about 90-95% or more homologous. A substantially homologous aminoacid sequence, according to the present invention, will be encoded by anucleic acid sequence hybridizing to the nucleic acid sequence, orportion thereof, of the sequence shown in SEQ ID NO:2 under stringentconditions as more fully described below.

[0078] To determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, even more preferably at least 60%,and even more preferably at least 70%, 80%, or 90% of the length of thereference sequence (e.g., when aligning a second sequence to the aminoacid sequence herein having 372 amino acid residues, at least 111,preferably at least 149, more preferably at least 186, even morepreferably at least 223, and even more preferably at least 260, 297,335, and 372 amino acid residues are aligned). The amino acid residuesor nucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

[0079] The invention also encompasses polypeptides having a lower degreeof identity but having sufficient similarity so as to perform one ormore of the same functions performed by the ubiquitin protease.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Conservativesubstitutions are likely to be phenotypically silent. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu, and Ile; interchange of thehydroxyl residues Ser and Thr, exchange of the acidic residues Asp andGlu, substitution between the amide residues Asn and Gln, exchange ofthe basic residues Lys and Arg and replacements among the aromaticresidues Phe, Tyr. Guidance concerning which amino acid changes arelikely to be phenotypically silent are found in Bowie et al., Science247:1306-1310 (1990). TABLE 1 Conservative Amino Acid Substitutions.Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic LeucineIsoleucine Valine Polar Glutamine Asparagine Basic Arginine LysineHistidine Acidic Aspartic Acid Glutamic Acid Small Alanine SerineThreonine Methionine Glycine

[0080] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

[0081] A preferred, non-limiting example of such a mathematicalalgorithm is described in Karlin et al. (1993) Proc. Natl. Acad. Sci.USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs (version 2.0) as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,NBLAST) can be used. In one embodiment, parameters for sequencecomparison can be set at score=100, wordlength=12, or can be varied(e.g., W=5 or W=20).

[0082] In a preferred embodiment, the percent identity between two aminoacid sequences is determined using the Needleman et al. (1970) (J. Mol.Biol. 48:444-453) algorithm which has been incorporated into the GAPprogram in the GCG software package using either a BLOSUM 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package (Devereuxet al. (1984) Nucleic Acids Res. 12(1):387) using a NWSgapdna.CMP matrixand a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,3, 4, 5, or 6.

[0083] Another preferred, non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, CABIOS (1989). Such an algorithm is incorporated intothe ALIGN program (version 2.0) which is part of the CGC sequencealignment software package. When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used. Additionalalgorithms for sequence analysis are known in the art and includeADVANCE and ADAM as described in Torellis et al. (1994) Comput. Appl.Biosci. 10:3-5; and FASTA described in Pearson et al. (1988) PNAS85:2444-8.

[0084] A variant polypeptide can differ in amino acid sequence by one ormore substitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these.

[0085] Variant polypeptides can be fully ftmctional or can lack functionin one or more activities. Thus, in the present case, variations canaffect the function, for example, of ubiquitin binding, ubiquitinrecognition, interaction with ubiquitinated substrate protein, such asbinding or proteolysis, subunit interaction, particularly within theproteasome, activation or binding by ATP, developmental expression,temporal expression, tissue-specific expression, interacting withcellular components, such as transcriptional regulatory factors, andparticularly trans-acting transcriptional regulatory factors,proteolytic cleavage of peptide bonds in polyubiquitin and peptide bondsbetween ubiquitin or polyubiquitin and substrate protein, andproteolytic cleavage of peptide bonds between ubiquitin or polyubiquitinand a peptide or amino acid.

[0086] Fully functional variants typically contain only conservativevariation or variation in non-critical residues or in non-criticalregions. Functional variants can also contain substitution of similaramino acids, which results in no change or an insignificant change infunction. Alternatively, such substitutions may positively or negativelyaffect function to some degree.

[0087] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0088] As indicated, variants can be naturally-occurring or can be madeby recombinant means or chemical synthesis to provide useful and novelcharacteristics for the ubiquitin protease polypeptide. This includespreventing immunogenicity from pharmaceutical formulations by preventingprotein aggregation.

[0089] Useful variations further include alteration of catalyticactivity. For example, one embodiment involves a variation at thebinding site that results in binding but not hydrolysis, or slowerhydrolysis, of the peptide bond. A further useful variation results inan increased rate of hydrolysis of the peptide bond. A further usefulvariation at the same site can result in higher or lower affinity forsubstrate. Useful variations also include changes that provide foraffinity for a different ubiquitinated substrate protein than thatnormally recognized. Other useful variations involving alteredrecognition affect recognition of the type of substrate normallyrecognized. For example, one variation could result in recognition ofubiquitinated intact substrate but not of substrate remnants, such asubiquitinated amino acid or peptide that are proteolysis products thatresult from the hydrolysis of the intact ubiquitinated substrate.Alternatively, the protease could be varied so that one or more of theremnant products is recognized but not the intact protein substrate.Another variation would affect the ability of the protease to rescue aubiquitinated protein. Thus, protein substrates that are normallyrescued from proteolysis would be subject to degradation. Further usefulvariations affect the ability of the protease to be induced byactivators, such as cytokines, including but not limited to, thosedisclosed herein. Another useful variation would affect the recognitionof ubiquitin substrate so that the enzyme could not recognize one ormore of a linear polyubiquitin, branched chain polyubiquitin, linearpolyubiquitinated substrate, or branched chain polyubiquitin substrate.Specific variations include truncation in which, for example, a HISdomain is deleted, the variation resulting in decrease or loss ofdeubiquitination activity. Another useful variation includes one thatprevents activation by ATP. Another useful variation provides a fusionprotein in which one or more domains or subregions are operationallyfused to one or more domains or subregions from another UBP or from aUCH. Specifically, a domain or subregion can be introduced that providesa rescue function to an enzyme not normally having this function or forrecognition of a specific substrate wherein recognition is not availableto the original enzyme. Other variations include those that affectubiquitin recognition or recognition of a ubiquitinated substrateprotein. Further variations could affect specific subunit interaction,particularly in the proteasome. Other variations would affectdevelopmental, temporal, or tissue-specific expression. Other variationswould affect the interaction with cellular components, such astranscriptional regulatory factors.

[0090] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al (1985) Science244:1081-1085). The latter procedure introduces single alanine mutationsat every residue in the molecule. The resulting mutant molecules arethen tested for biological activity, such as peptide hydrolysis in vitroor ubiquitin-dependent in vitro activity, such as proliferativeactivity, receptor-mediated signal transduction, and other cellularprocesses including, but not limited, those disclosed herein that are afunction of the ubiquitin system. Sites that are critical for binding orrecognition can also be determined by structural analysis such ascrystallization, nuclear magnetic resonance or photoaffinity labeling(Smith et al. (1992) J. Mol. Biol. 224:899-904; de Vos et al. (1992)Science 255:306-312).

[0091] The assays for deubiquitinating enzyme activity are well known inthe art and can be found, for example, in Zhu et al. (1997) Journal ofBiological Chemistry 272:51-57, Mitch et al. (1999)

[0092] American Journal of Physiology 276:C1132-C1138, Liu et al. (1999)Molecular and Cell Biology 19:3029-3038, and such as those cited invarious reviews, for example, Ciechanover et al. (1994) The FASEBJournal 8:182-192, Chiechanover (1994) Biol. Chem. Hoppe-Seyler375:565-581, Hershko et al. (1998) Annual Review of Biochemistry67:425-479, Swartz (1999) Annual Review of Medicine 50:57-74,Ciechanover (1998) EMBO Journal 17:7151-7160, and D'Andrea et al. (1998)Critical Reviews in Biochemistry and Molecular Biology 33:337-352. Theseassays include, but are not limited to, the disappearance of substrate,including decrease in the amount of polyubiquitin or ubiquitinatedsubstrate protein or protein remnant, appearance of intermediate and endproducts, such as appearance of free ubiquitin monomers, general proteinturnover, specific protein turnover, ubiquitin binding, binding toubiquitinated substrate protein, subunit interaction, interaction withATP, interaction with cellular components such as trans-actingregulatory factors, stabilization of specific proteins, and the like.

[0093] Substantial homology can be to the entire nucleic acid or aminoacid sequence or to fragments of these sequences.

[0094] The invention thus also includes polypeptide fragments of theubiquitin protease. Fragments can be derived from the amino acidsequence shown in SEQ ID NO:1. However, the invention also encompassesfragments of the variants of the ubiquitin proteases as describedherein.

[0095] The fragments to which the invention pertains, however, are notto be construed as encompassing fragments that may be disclosed prior tothe present invention.

[0096] Accordingly, a fragment can comprise at least about 10, 15, 20,25, 30, 35, 40, 45, 50 or more contiguous amino acids. Fragments canretain one or more of the biological activities of the protein, forexample the ability to bind to ubiquitin or hydrolyze peptide bonds, aswell as fragments that can be used as an immunogen to generate ubiquitinprotease antibodies.

[0097] Biologically active fragments (peptides which are, for example,5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more aminoacids in length) can comprise a domain or motif, e.g., catalytic site,UBP or UCH signature, membrane-associated regions and sites forglycosylation, cAMP and cGMP-dependent protein kinase phosphorylation,protein kinase C phosphorylation, casein kinase II phosphorylation,tyrosine kinase phosphorylation, N-myristoylation, and amidation.Further possible fragments include the catalytic site or domainincluding the cysteine or histidine boxes as shown in FIG. 1, ubiquitinrecognition sites, ubiquitin binding sites, sites important for subunitinteraction, and sites important for carrying out the other functions ofthe protease as described herein.

[0098] Such domains or motifs can be identified by means of routinecomputerized homology searching procedures.

[0099] Fragments, for example, can extend in one or both directions fromthe functional site to encompass 5, 10, 15, 20, 30, 40, 50, or up to 100amino acids. Further, fragments can include sub-fragments of thespecific domains mentioned above, which sub-fragments retain thefunction of the domain from which they are derived.

[0100] These regions can be identified by well-known methods involvingcomputerized homology analysis.

[0101] The invention also provides fragments with immunogenicproperties. These contain an epitope-bearing portion of the ubiquitinprotease and variants. These epitope-bearing peptides are useful toraise antibodies that bind specifically to a ubiquitin proteasepolypeptide or region or fragment. These peptides can contain at least10, 12, at least 14, or between at least about 15 to about 30 aminoacids.

[0102] Non-limiting examples of antigenic polypeptides that can be usedto generate antibodies include but are not limited to peptides derivedfrom an extracellular site. Regions having a high antigenicity index areshown in FIG. 3. However, intracellularly-made antibodies(“intrabodies”) are also encompassed, which would recognizeintracellular peptide regions.

[0103] The epitope-bearing ubiquitin protease polypeptides may beproduced by any conventional means (Houghten, R. A. (1985) Proc. Natl.Acad. Sci. USA 82:5131-5135). Simultaneous multiple peptide synthesis isdescribed in U.S. Pat. No. 4,631,211.

[0104] Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Further, severalfragments can be comprised within a single larger polypeptide. In oneembodiment a fragment designed for expression in a host can haveheterologous pre- and pro-polypeptide regions fused to the aminoterminus of the ubiquitin protease fragment and an additional regionfused to the carboxyl terminus of the fragment.

[0105] The invention thus provides chimeric or fusion proteins. Thesecomprise a ubiquitin protease peptide sequence operatively linked to aheterologous peptide having an amino acid sequence not substantiallyhomologous to the ubiquitin protease. “Operatively linked” indicatesthat the ubiquitin protease peptide and the heterologous peptide arefused in-frame. The heterologous peptide can be fused to the N-terminusor C-terminus of the ubiquitin protease or can be internally located.

[0106] In one embodiment the fusion protein does not affect ubiquitinprotease function per se. For example, the fusion protein can be aGST-fusion protein in which the ubiquitin protease sequences are fusedto the C-terminus of the GST sequences. Other types of fusion proteinsinclude, but are not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL-4 fusions, poly-Hisfusions and Ig fusions. Such fusion proteins, particularly poly-Hisfusions, can facilitate the purification of recombinant ubiquitinprotease. In certain host cells (e.g., mammalian host cells), expressionand/or secretion of a protein can be increased by using a heterologoussignal sequence. Therefore, in another embodiment, the fusion proteincontains a heterologous signal sequence at its N-terminus.

[0107] EP-A-O 464 533 discloses fusion proteins comprising variousportions of immunoglobulin constant regions. The Fc is useful in therapyand diagnosis and thus results, for example, in improved pharmacokineticproperties (EP-A 0232 262). In drug discovery, for example, humanproteins have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists (Bennett et al.(1995) J Mol. Recog. 8:52-58 (1995) and Johanson et al. J Biol. Chem.270:9459-9471). Thus, this invention also encompasses soluble fusionproteins containing a ubiquitin protease polypeptide and variousportions of the constant regions of heavy or light chains ofimmunoglobulins of various subclass (IgG, IgM, IgA, IgE). Preferred asimmunoglobulin is the constant part of the heavy chain of human IgG,particularly IgG1, where fusion takes place at the hinge region. Forsome uses it is desirable to remove the Fc after the fusion protein hasbeen used for its intended purpose, for example when the fusion proteinis to be used as antigen for immunizations. In a particular embodiment,the Fc part can be removed in a simple way by a cleavage sequence, whichis also incorporated and can be cleaved with factor Xa.

[0108] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al. (1992) Current Protocols in Molecular Biology). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A ubiquitin protease-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the ubiquitin protease.

[0109] Another form of fusion protein is one that directly affectsubiquitin protease functions. Accordingly, a ubiquitin proteasepolypeptide is encompassed by the present invention in which one or moreof the ubiquitin protease domains (or parts thereof) has been replacedby homologous domains (or parts thereof) from another UBP or UCHspecies. Accordingly, various permutations are possible. One or morefunctional sites as disclosed herein from the specifically disclosedprotease can be replaced by one or more functional sites from acorresponding UBP family member or from a UCH family member. Thus,chimeric ubiquitin proteases can be formed in which one or more of thenative domains or subregions has been replaced by another.

[0110] Additionally, chimeric ubiquitin protease proteins can beproduced in which one or more functional sites is derived from adifferent ubiquitin protease family. It is understood however that sitescould be derived from ubiquitin protease families that occur in themammalian genome but which have not yet been discovered orcharacterized. Such sites include but are not limited to any of thefunctional sites disclosed herein.

[0111] The isolated ubiquitin proteases can be purified from cells thatnaturally express it, such as from thymus, testes, brain, breast,skeletal muscle, liver, prostate, thyroid, ovary, fetal kidney, fetalheart, fetal liver, liver metastases derived from colon, and malignantlung and breast tissue, especially purified from cells that have beenaltered to express it (recombinant), or synthesized using known proteinsynthesis methods.

[0112] In one embodiment, the protein is produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding the ubiquitinprotease polypeptide is cloned into an expression vector, the expressionvector introduced into a host cell and the protein expressed in the hostcell. The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques.Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally-occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in polypeptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art.

[0113] Accordingly, the polypeptides also encompass derivatives oranalogs in which a substituted amino acid residue is not one encoded bythe genetic code, in which a substituent group is included, in which themature polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or in which the additional amino acids are fused to the maturepolypeptide, such as a leader or secretory sequence or a sequence forpurification of the mature polypeptide or a pro-protein sequence.

[0114] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0115] Such modifications are well-known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2nded., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (1990) Meth.Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N.Y. Acad. Sci.663:48-62).

[0116] As is also well known, polypeptides are not always entirelylinear. For instance, polypeptides may be branched as a result ofubiquitination, and they may be circular, with or without branching,generally as a result of post-translation events, including naturalprocessing events and events brought about by human manipulation whichdo not occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translational natural processesand by synthetic methods.

[0117] Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. Blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally-occurring andsynthetic polypeptides. For instance, the aminoterminal residue ofpolypeptides made in E. coli, prior to proteolytic processing, almostinvariably will be N-formylmethionine.

[0118] The modifications can be a function of how the protein is made.For recombinant polypeptides, for example, the modifications will bedetermined by the host cell posttranslational modification capacity andthe modification signals in the polypeptide amino acid sequence.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to efficiently express mammalian proteins havingnative patterns of glycosylation. Similar considerations apply to othermodifications.

[0119] The same type of modification may be present in the same orvarying degree at several sites in a given polypeptide. Also, a givenpolypeptide may contain more than one type of modification.

[0120] Polypeptide Uses

[0121] The protein sequences of the present invention can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score 100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the proteins of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used.

[0122] The ubiquitin protease polypeptides are useful for producingantibodies specific for the ubiquitin protease, regions, or fragments.Regions having a high antigenicity index score are shown in FIG. 3.

[0123] The ubiquitin protease polypeptides are useful for biologicalassays related to ubiquitin protease function. Such assays involve anyof the known functions or activities or properties useful for diagnosisand treatment of ubiquitin- or ubiquitin protease-related conditions.Potential assays have been disclosed herein and generically includedisappearance of substrate, appearance of end product, and general orspecific protein turnover.

[0124] The ubiquitin protease polypeptides are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the ubiquitin protease,as a biopsy or expanded in cell culture. In one embodiment, however,cell-based assays involve recombinant host cells expressing theubiquitin protease.

[0125] Determining the ability of the test compound to interact with theubiquitin protease can also comprise determining the ability of the testcompound to preferentially bind to the polypeptide as compared to theability of a known binding molecule (e.g., ubiquitin) to bind to thepolypeptide.

[0126] The polypeptides can be used to identify compounds that modulateubiquitin protease activity. Such compounds, for example, can increaseor decrease affinity for polyubiquitin, either linear or branched chain,ubiquitinated protein substrate, or ubiquitinated protein substrateremnants. Such compounds could also, for example, increase or decreasethe rate of binding to these components. Such compounds could alsocompete with these components for binding to the ubiquitin protease ordisplace these components bound to the ubiquitin protease. Suchcompounds could also affect interaction with other components, such asATP, other subunits, for example, in the 19S complex, andtranscriptional regulatory factors. It is understood, therefore, thatsuch compounds can be identified not only by means of ubiquitin, but bymeans of any of the components that functionally interact with thedisclosed protease. This includes, but is not limited to, any of thosecomponents disclosed herein.

[0127] Both ubiquitin protease and appropriate variants and fragmentscan be used in high-throughput screens to assay candidate compounds forthe ability to bind to the ubiquitin protease. These compounds can befurther screened against a functional ubiquitin protease to determinethe effect of the compound on the ubiquitin protease activity. Compoundscan be identified that activate (agonist) or inactivate (antagonist) theubiquitin protease to a desired degree. Modulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject.

[0128] The ubiquitin protease polypeptides can be used to screen acompound for the ability to stimulate or inhibit interaction between theubiquitin protease protein and a target molecule that normally interactswith the ubiquitin protease protein. The target can be ubiquitin,ubiquitinated substrate, or polyubiquitin or another component of thepathway with which the ubiquitin protease protein normally interacts(for example, ATP). The assay includes the steps of combining theubiquitin protease protein with a candidate compound under conditionsthat allow the ubiquitin protease protein or fragment to interact withthe target molecule, and to detect the formation of a complex betweenthe ubiquitin protease protein and the target or to detect thebiochemical consequence of the interaction with the ubiquitin proteaseand the target. Any of the associated effects of protease function canbe assayed. This includes the production of hydrolysis products, such asfree terminal peptide substrate, free terminal amino acid from thehydrolyzed substrate, free ubiquitin, lower molecular weight species ofhydrolyzed polyubiquitin, released intact substrate protein resultingfrom rescue from proteolysis, free polyubiquitin formed from hydrolysisof the polyubiquitin from intact substrate, and substrate remnants, suchas amino acids and peptides produced from proteolysis of the substrateprotein, and biological endpoints of the pathway.

[0129] Determining the ability of the ubiquitin protease to bind to atarget molecule can also be accomplished using a technology such asreal-time Bimolecular Interaction Analysis (BIA). Sjolander et al.(1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin.Struct. Biol. 5:699-705. As used herein, “BIA” is a technology forstudying biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore™). Changes in the optical phenomenonsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

[0130] The test compounds of the present invention can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to polypeptide libraries, whilethe other four approaches are applicable to polypeptide, non-peptideoligomer or small molecule libraries of compounds (Lam, K. S. (1997)Anticancer Drug Des. 12:145).

[0131] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in DeWitt et al. (1993) Proc. Natl.Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries ofcompounds maybe presented in solution (e.g., Houghten (1992)Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84),chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No.5,223,409), spores (Ladner U.S. patent '409), plasmids (Cull et al.(1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott andSmith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 97:6378-6382); (Felici(1991) J. Mol. Biol. 222:301-310); (Ladner supra).

[0132] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al. (1991) Nature 354:82-84;Houghten et al. (1991) Nature 354:84-86) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal. (1993) Cell 72:767-778); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0133] One candidate compound is a soluble full-length ubiquitinprotease or fragment that competes for substrate binding. Othercandidate compounds include mutant ubiquitin proteases or appropriatefragments containing mutations that affect ubiquitin protease functionand compete for substrate. Accordingly, a fragment that competes forsubstrate, for example with a higher affinity, or a fragment that bindssubstrate but does not hydrolyze the peptide bond, is encompassed by theinvention.

[0134] Other candidate compounds include ubiquitinated protein orprotein analog that binds to the protease but is not released orreleased slowly. Other candidate compounds include analogs of the othernatural substrates, such as substrate remnants that bind to but are notreleased or released more slowly. Further candidate compounds includeactivators of the proteases such as cytokines, including but not limitedto, those disclosed herein.

[0135] The invention provides other end points to identify compoundsthat modulate (stimulate or inhibit) ubiquitin protease activity. Theassays typically involve an assay of events in the pathway that indicateubiquitin protease activity. This can include cellular events thatresult from deubiquitination, such as cell cycle progression, programmedcell death, growth factor-mediated signal transduction, or any of thecellular processes including, but not limited to, those disclosed hereinas resulting from deubiquitination. Specific phenotypes include changesin stress response, DNA replication, receptor internalization, cellulartransformation or reversal of transformation, and transcriptionalsilencing.

[0136] Assays are based on the multiple cellular functions ofdeubiquitinating enzymes. These enzymes act at various different levelsin the regulation of protein ubiquitination. A deubiquitinating enzymecan degrade a linear polyubiquitin chain into monomeric ubiquitinmolecules. Deubiquitinating enzymes, such as isopeptidase-T, can degradea branched multiubiquitin chain into monomeric ubiquitin molecules.Deubiquitinating enzymes can remove ubiquitin from aubiquitin-conjugated target protein. The deubiquitinating enzyme, suchas FAF or PA700 isopeptidase, can remove polyubiquitin from aubiquitinated target protein, and thereby rescue the target fromdegradation by the 26S proteasome. Deubiquitinating enzymes such asDoa-4 can remove polyubiquitin from proteasome degradation products. Theresult of all of these is to regulate the cellular pool of freemonomeric ubiquitin. Accordingly, assays can be based on detection ofany of the products produced by hydrolysis/deubiquitination.

[0137] Further, the expression of genes that are up- or down-regulatedby action of the ubiquitin protease can be assayed. In one embodiment,the regulatory region of such genes can be operably linked to a markerthat is easily detectable, such as luciferase.

[0138] Accordingly, any of the biological or biochemical functionsmediated by the ubiquitin protease can be used as an endpoint assay.These include all of the biochemical or biochemical/biological eventsdescribed herein, in the references cited herein, incorporated byreference for these endpoint assay targets, and other functions known tothose of ordinary skill in the art.

[0139] Binding and/or activating compounds can also be screened by usingchimeric ubiquitin protease proteins in which one or more domains,sites, and the like, as disclosed herein, or parts thereof, can bereplaced by their heterologous counterparts derived from other ubiquitinproteases. For example, a recognition or binding region can be used thatinteracts with different substrate specificity and/or affinity than thenative ubiquitin protease. Accordingly, a different set of pathwaycomponents is available as an end-point assay for activation. Further,sites that are responsible for developmental, temporal, or tissuespecificity can be replaced by heterologous sites such that the proteasecan be detected under conditions of specific developmental, temporal, ortissue-specific expression.

[0140] The ubiquitin protease polypeptides are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the ubiquitin protease. Thus, a compound is exposedto a ubiquitin protease polypeptide under conditions that allow thecompound to bind to or to otherwise interact with the polypeptide.Soluble ubiquitin protease polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble ubiquitin proteasepolypeptide, it decreases the amount of complex formed or activity fromthe ubiquitin protease target. This type of assay is particularly usefulin cases in which compounds are sought that interact with specificregions of the ubiquitin protease. Thus, the soluble polypeptide thatcompetes with the target ubiquitin protease region is designed tocontain peptide sequences corresponding to the region of interest.

[0141] Another type of competition-binding assay can be used to discovercompounds that interact with specific functional sites. As an example,ubiquitin and a candidate compound can be added to a sample of theubiquitin protease. Compounds that interact with the ubiquitin proteaseat the same site as ubiquitin will reduce the amount of complex formedbetween the ubiquitin protease and ubiquitin. Accordingly, it ispossible to discover a compound that specifically prevents interactionbetween the ubiquitin protease and ubiquitin. Another example involvesadding a candidate compound to a sample of ubiquitin protease andpolyubiquitin. A compound that competes with polyubiquitin will reducethe amount of hydrolysis or binding of the polyubiquitin to theubiquitin protease. Accordingly, compounds can be discovered thatdirectly interact with the ubiquitin protease and compete withpolyubiquitin. Such assays can involve any other component thatinteracts with the ubiquitin protease, such as ubiquitinated substrateprotein, ubiquitinated substrate remnants, and cellular components withwhich the protease interacts such as transcriptional regulatory factors.

[0142] To perform cell free drug screening assays, it is desirable toimmobilize either the ubiquitin protease, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

[0143] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase/ubiquitin protease fusionproteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the cell lysates (e.g., ³⁵S-labeled) andthe candidate compound, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads are washed to remove anyunbound label, and the matrix immobilized and radiolabel determineddirectly, or in the supernatant after the complexes is dissociated.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of ubiquitin protease-bindingprotein found in the bead fraction quantitated from the gel usingstandard electrophoretic techniques. For example, either the polypeptideor its target molecule can be immobilized utilizing conjugation ofbiotin and streptavidin using techniques well known in the art.Alternatively, antibodies reactive with the protein but which do notinterfere with binding of the protein to its target molecule can bederivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a ubiquitinprotease-binding target component, such as ubiquitin, polyubiquitin,ubiquitinated substrate protein, ubiquitinated substrate proteinremnant, or ubiquitinated remnant amino acid, and a candidate compoundare incubated in the ubiquitin protease-presenting wells and the amountof complex trapped in the well can be quantitated. Methods for detectingsuch complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the ubiquitin protease target molecule, orwhich are reactive with ubiquitin protease and compete with the targetmolecule; as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the target molecule.

[0144] Modulators of ubiquitin protease activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the ubiquitin protease pathway, by treating cellsthat express the ubiquitin protease, including but not limited to, thoseshown in FIGS. 6, 7, and 8 and especially liver, breast, brain, andtestes. In one embodiment, the cells treated are lung or breast cancercells. In another embodiment of the invention the cells that are treatedare colon metastases to the liver. These methods of treatment includethe steps of administering the modulators of ubiquitin protease activityin a pharmaceutical composition as described herein, to a subject inneed of such treatment.

[0145] Disorders involving the liver include, but are not limited to,hepatic injury; jaundice and cholestasis, such as bilirubin and bileformation; hepatic failure and cirrhosis, such as cirrhosis, portalhypertension, including ascites, portosystemic shunts, and splenomegaly;infectious disorders, such as viral hepatitis, including hepatitis A-Einfection and infection by other hepatitis viruses, clinicopathologicsyndromes, such as the carrier state, asymptomatic infection, acuteviral hepatitis, chronic viral hepatitis, and fulminant hepatitis;autoimmune hepatitis; drug- and toxin-induced liver disease, such asalcoholic liver disease; inborn errors of metabolism and pediatric liverdisease, such as hemochromatosis, Wilson disease, a₁-antitrypsindeficiency, and neonatal hepatitis; intrahepatic biliary tract disease,such as secondary biliary cirrhosis, primary biliary cirrhosis, primarysclerosing cholangitis, and anomalies of the biliary tree; circulatorydisorders, such as impaired blood flow into the liver, including hepaticartery compromise and portal vein obstruction and thrombosis, impairedblood flow through the liver, including passive congestion andcentrilobular necrosis and peliosis hepatis, hepatic vein outflowobstruction, including hepatic vein thrombosis (Budd-Chiari syndrome)and veno-occlusive disease; hepatic disease associated with pregnancy,such as preeclampsia and eclampsia, acute fatty liver of pregnancy, andintrehepatic cholestasis of pregnancy; hepatic complications of organ orbone marrow transplantation, such as drug toxicity after bone marrowtransplantation, graft-versus-host disease and liver rejection, andnonimmunologic damage to liver allografts; tumors and tumorousconditions, such as nodular hyperplasias, adenomas, and malignanttumors, including primary carcinoma of the liver and metastatic tumors.

[0146] Disorders involving the brain include, but are limited to,disorders involving neurons, and disorders involving glia, such asastrocytes, oligodendrocytes, ependymal cells, and microglia; cerebraledema, raised intracranial pressure and herniation, and hydrocephalus;malformations and developmental diseases, such as neural tube defects,forebrain anomalies, posterior fossa anomalies, and syringomyelia andhydromyelia; perinatal brain injury; cerebrovascular diseases, such asthose related to hypoxia, ischemia, and infarction, includinghypotension, hypoperfusion, and low-flow states—global cerebral ischemiaand focal cerebral ischemia—infarction from obstruction of local bloodsupply, intracranial hemorrhage, including intracerebral(intraparenchymal) hemorrhage, subarachnoid hemorrhage and rupturedberry aneurysms, and vascular malformations, hypertensivecerebrovascular disease, including lacunar infarcts, slit hemorrhages,and hypertensive encephalopathy; infections, such as acute meningitis,including acute pyogenic (bacterial) meningitis and acute aseptic(viral) meningitis, acute focal suppurative infections, including brainabscess, subdural empyema, and extradural abscess, chronic bacterialmeningoencephalitis, including tuberculosis and mycobacterioses,neurosyphilis, and neuroborreliosis (Lyme disease), viralmeningoencephalitis, including arthropod-bome (Arbo) viral encephalitis,Herpes simplex virus Type 1, Herpes simplex virus Type 2,Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis,rabies, and human immunodeficiency virus 1, including HIV-1meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degeneration,multiple system atrophy, including striatonigral degeneration,Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntingtondisease; spinocerebellar degenerations, including spinocerebellarataxias, including Friedreich ataxia, and ataxia-telanglectasia,degenerative diseases affecting motor neurons, including amyotrophiclateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

[0147] Disorders involving the heart, include but are not limited to,heart failure, including but not limited to, cardiac hypertrophy,left-sided heart failure, and right-sided heart failure; ischemic heartdisease, including but not limited to angina pectoris, myocardialinfarction, chronic ischemic heart disease, and sudden cardiac death;hypertensive heart disease, including but not limited to, systemic(left-sided) hypertensive heart disease and pulmonary (right-sided)hypertensive heart disease; valvular heart disease, including but notlimited to, valvular degeneration caused by calcification, such ascalcific aortic stenosis, calcification of a congenitally bicuspidaortic valve, and mitral annular calcification, and myxomatousdegeneration of the mitral valve (mitral valve prolapse), rheumaticfever and rheumatic heart disease, infective endocarditis, andnoninfected vegetations, such as nonbacterial thrombotic endocarditisand endocarditis of systemic lupus erythematosus (Libman-Sacks disease),carcinoid heart disease, and complications of artificial valves;myocardial disease, including but not limited to dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, andmyocarditis; pericardial disease, including but not limited to,pericardial effusion and hemopericardium and pericarditis, includingacute pericarditis and healed pericarditis, and rheumatoid heartdisease; neoplastic heart disease, including but not limited to, primarycardiac tumors, such as myxoma, lipoma, papillary fibroelastoma,rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms;congenital heart disease, including but not limited to, left-to-rightshunts—late cyanosis, such as atrial septal defect, ventricular septaldefect, patent ductus arteriosus, and atrioventricular septal defect,right-to-left shunts—early cyanosis, such as tetralogy of fallot,transposition of great arteries, truncus arteriosus, tricuspid atresia,and total anomalous pulmonary venous connection, obstructive congenitalanomalies, such as coarctation of aorta, pulmonary stenosis and atresia,and aortic stenosis and atresia, and disorders involving cardiactransplantation.

[0148] Disorders involving the thymus include developmental disorders,such as DiGeorge syndrome with thymic hypoplasia or aplasia; thymiccysts; thymic hypoplasia, which involves the appearance of lymphoidfollicles within the thymus, creating thymic follicular hyperplasia; andthymomas, including germ cell tumors, lynphomas, Hodgkin disease, andcarcinoids. Thymomas can include benign or encapsulated thymoma, andmalignant thymoma Type I (invasive thymoma) or Type II, designatedthymic carcinoma.

[0149] Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease and simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schönlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including, but not limited to, acutetubular necrosis and tubulointerstitial nephritis, including but notlimited to, pyelonephritis and urinary tract infection, acutepyelonephritis, chronic pyelonephritis and reflux nephropathy,tubulointerstitial nephritis induced by drugs and toxins, including butnot limited to, acute drug-induced interstitial nephritis, analgesicabuse nephropathy, and nephropathy associated with nonsteroidalanti-inflammatory drugs, and other tubulointerstitial diseasesincluding, but not limited to, urate nephropathy, hypercalcemia andnephrocalcinosis, and multiple myeloma; diseases of blood vesselsincluding, including but not limited to, benign nephrosclerosis,malignant hypertension and accelerated nephrosclerosis, renal arterystenosis, and thrombotic microangiopathies, including, but not limitedto, classic (childhood) hemolytic-uremic syndrome, adulthemolytic-uremic syndrome/thrombotic thrombocytopenic purpura,idiopathic HUS/TTP, and other vascular disorders including, but notlimited to, atherosclerotic ischemic renal disease, atheroembolic renaldisease, sickle cell disease nephropathy, diffuse cortical necrosis, andrenal infarcts; urinary tract obstruction (obstructive uropathy);urolithiasis (renal calculi, stones); and tumors of the kidneyincluding, but not limited to, benign tumors, such as renal papillaryadenoma, renal fibroma or hamartoma (renomedullary interstitial celltumor), angiomyolipoma, and oncocytoma, and malignant tumors, includingrenal cell carcinoma (hypemephroma, adenocarcinoma of kidney), whichincludes urothelial carcinomas of renal pelvis.

[0150] Disorders of the breast include, but are not limited to,disorders of development; inflammations, including but not limited to,acute mastitis, periductal mastitis (recurrent subareolar abscess,squamous metaplasia of lactiferous ducts), mammary duct ectasia, fatnecrosis, granulomatous mastitis, and pathologies associated withsilicone breast implants; fibrocystic changes; proliferative breastdisease including, but not limited to, epithelial hyperplasia,sclerosing adenosis, and small duct papillomas; tumors including, butnot limited to, stromal tumors such as fibroadenoma, phyllodes tumor,and sarcomas, and epithelial tumors, such as large duct papilloma;carcinoma of the breast including in situ (noninvasive) carcinoma thatincludes ductal carcinoma in situ (including Paget's disease) andlobular carcinoma in situ, and invasive (infiltrating) carcinomaincluding, but not limited to, invasive ductal carcinoma, no specialtype, invasive lobular carcinoma, medullary carcinoma, colloid(mucinous) carcinoma, tubular carcinoma, and invasive papillarycarcinoma, and miscellaneous malignant neoplasms. Disorders in the malebreast include, but are not limited to, gynecomastia and carcinoma.

[0151] Disorders involving the testis and epididymis include, but arenot limited to, congenital anomalies such as cryptorchidism, regressivechanges such as atrophy, inflammations such as nonspecific epididymitisand orchitis, granulomatous (autoimmune) orchitis, and specificinflammations including, but not limited to, gonorrhea, mumps,tuberculosis, and syphilis, vascular disturbances including torsion,testicular tumors including germ cell tumors that include, but are notlimited to, seminoma, spermatocytic seminoma, embryonal carcinoma, yolksac tumor, choriocarcinoma, teratoma, and mixed tumors, tumors of sexcord-gonadal stroma including, but not limited to, Leydig (interstitial)cell tumors and Sertoli cell tumors (androblastoma), and testicularlymphoma, and miscellaneous lesions of tunica vaginalis.

[0152] Disorders involving the prostate include, but are not limited to,inflammations, benign enlargement, for example, nodular hyperplasia(benign prostatic hypertrophy or hyperplasia), and tumors such ascarcinoma.

[0153] Disorders involving the thyroid include, but are not limited to,hyperthyroidism; hypothyroidism including, but not limited to, cretinismand myxedema; thyroiditis including, but not limited to, hashimotothyroiditis, subacute (granulomatous) thyroiditis, and subacutelymphocytic (painless) thyroiditis; Graves disease; diffuse andmultinodular goiter including, but not limited to, diffuse nontoxic(simple) goiter and multinodular goiter; neoplasms of the thyroidincluding, but not limited to, adenomas, other benign tumors, andcarcinomas, which include, but are not limited to, papillary carcinoma,follicular carcinoma, medullary carcinoma, and anaplastic carcinoma; andcogenital anomalies.

[0154] Disorders involving the skeletal muscle include tumors, such asrhabdomyosarcoma.

[0155] The ubiquitin protease polypeptides are thus useful for treatinga ubiquitin protease-associated disorder characterized by aberrantexpression or activity of a ubiquitin protease. The polypeptides canalso be useful for treating a disorder characterized by excessiveamounts of polyubiquitin or ubiquitinated substrate/remnant/amino acid.In one embodiment, the method involves administering an agent (e.g., anagent identified by a screening assay described herein), or combinationof agents that modulates (e.g., upregulates or downregulates) expressionor activity of the protein. In another embodiment, the method involvesadministering the ubiquitin protease as therapy to compensate forreduced or aberrant expression or activity of the protein.

[0156] Methods for treatment include but are not limited to the use ofsoluble ubiquitin protease or fragments of the ubiquitin proteaseprotein that compete for substrates including those disclosed herein.These ubiquitin proteases or fragments can have a higher affinity forthe target so as to provide effective competition.

[0157] Stimulation of activity is desirable in situations in which theprotein is abnormally downregulated and/or in which increased activityis likely to have a beneficial effect. Likewise, inhibition of activityis desirable in situations in which the protein is abnormallyupregulated and/or in which decreased activity is likely to have abeneficial effect. In one example of such a situation, a subject has adisorder characterized by aberrant development or cellulardifferentiation. In another example, the subject has a proliferativedisease (e.g., cancer) or a disorder characterized by an aberranthematopoietic response. In another example, it is desirable to achievetissue regeneration in a subject (e.g., where a subject has undergonebrain or spinal cord injury and it is desirable to regenerate neuronaltissue in a regulated manner).

[0158] In yet another aspect of the invention, the proteins of theinvention can be used as “bait proteins” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO 94/10300), to identifyother proteins (captured proteins) which bind to or interact with theproteins of the invention and modulate their activity.

[0159] The ubiquitin protease polypeptides also are useful to provide atarget for diagnosing a disease or predisposition to disease mediated bythe ubiquitin protease, including, but not limited to, diseasesinvolving tissues in which the ubiquitin proteases are expressed asdisclosed herein, such as in breast cancer. Accordingly, methods areprovided for detecting the presence, or levels of, the ubiquitinprotease in a cell, tissue, or organism. The method involves contactinga biological sample with a compound capable of interacting with theubiquitin protease such that the interaction can be detected.

[0160] The polypeptides are also useful for treating a disordercharacterized by reduced amounts of these components. Thus, increasingor decreasing the activity of the protease is beneficial to treatment.The polypeptides are also useful to provide a target for diagnosing adisease characterized by excessive substrate or reduced levels ofsubstrate. Accordingly, where substrate is excessive, use of theprotease polypeptides can provide a diagnostic assay. Furthermore, forexample, proteases having reduced activity can be used to diagnoseconditions in which reduced substrate is responsible for the disorder.

[0161] One agent for detecting ubiquitin protease is an antibody capableof selectively binding to ubiquitin protease. A biological sampleincludes tissues, cells and biological fluids isolated from a subject,as well as tissues, cells and fluids present within a subject.

[0162] The ubiquitin protease also provides a target for diagnosingactive disease, or predisposition to disease, in a patient having avariant ubiquitin protease. Thus, ubiquitin protease can be isolatedfrom a biological sample and assayed for the presence of a geneticmutation that results in an aberrant protein. This includes amino acidsubstitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered ubiquitin protease activity incell-based or cell-free assay, alteration in binding to or hydrolysis ofpolyubiquitin, binding to ubiquitinated substrate protein or hydrolysisof the ubiquitin from the protein, binding to ubiquitinated proteinremnant, including peptide or amino acid, and hydrolysis of theubiquitin from the remnant, general protein turnover, specific proteinturnover, antibody-binding pattern, altered isoelectric point, directamino acid sequencing, and any other of the known assay techniquesuseful for detecting mutations in a protein in general or in a ubiquitinprotease specifically, including assays discussed herein.

[0163] In vitro techniques for detection of ubiquitin protease includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. Alternatively, the proteincan be detected in vivo in a subject by introducing into the subject alabeled anti-ubiquitin protease antibody. For example, the antibody canbe labeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques. Particularlyuseful are methods, which detect the allelic variant of the ubiquitinprotease expressed in a subject, and methods, which detect fragments ofthe ubiquitin protease in a sample.

[0164] The ubiquitin protease polypeptides are also useful inpharmacogenomic analysis. Pharmacogenomics deal with clinicallysignificant hereditary variations in the response to drugs due toaltered drug disposition and abnormal action in affected persons. See,e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol.23(10-11):983-985, and Linder, M. W. (1997) Clin. Chem. 43(2):254-266.The clinical outcomes of these variations result in severe toxicity oftherapeutic drugs in certain individuals or therapeutic failure of drugsin certain individuals as a result of individual variation inmetabolism. Thus, the genotype of the individual can determine the way atherapeutic compound acts on the body or the way the body metabolizesthe compound. Further, the activity of drug metabolizing enzymes affectsboth the intensity and duration of drug action. Thus, thepharmacogenomics of the individual permit the selection of effectivecompounds and effective dosages of such compounds for prophylactic ortherapeutic treatment based on the individual's genotype. The discoveryof genetic polymorphisms in some drug metabolizing enzymes has explainedwhy some patients do not obtain the expected drug effects, show anexaggerated drug effect, or experience serious toxicity from standarddrug dosages. Polymorphisms can be expressed in the phenotype of theextensive metabolizer and the phenotype of the poor metabolizer.Accordingly, genetic polymorphism may lead to allelic protein variantsof the ubiquitin protease in which one or more of the ubiquitin proteasefunctions in one population is different from those in anotherpopulation. The polypeptides thus allow a target to ascertain a geneticpredisposition that can affect treatment modality. Thus, in aubiquitin-based treatment, polymorphism may give rise to catalyticregions that are more or less active. Accordingly, dosage wouldnecessarily be modified to maximize the therapeutic effect within agiven population containing the polymorphism. As an alternative togenotyping, specific polymorphic polypeptides could be identified.

[0165] The ubiquitin protease polypeptides are also useful formonitoring therapeutic effects during clinical trials and othertreatment. Thus, the therapeutic effectiveness of an agent that isdesigned to increase or decrease gene expression, protein levels orubiquitin protease activity can be monitored over the course oftreatment using the ubiquitin protease polypeptides as an end-pointtarget. The monitoring can be, for example, as follows: (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression or activity of the proteinin the pre-administration sample; (iii) obtaining one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the protein in the post-administrationsamples; (v) comparing the level of expression or activity of theprotein in the pre-administration sample with the protein in thepost-administration sample or samples; and (vi) increasing or decreasingthe administration of the agent to the subject accordingly.

[0166] Antibodies

[0167] The invention also provides antibodies that selectively bind tothe ubiquitin protease and its variants and fragments. An antibody isconsidered to selectively bind, even if it also binds to other proteinsthat are not substantially homologous with the ubiquitin protease. Theseother proteins share homology with a fragment or domain of the ubiquitinprotease. This conservation in specific regions gives rise to antibodiesthat bind to both proteins by virtue of the homologous sequence. In thiscase, it would be understood that antibody binding to the ubiquitinprotease is still selective.

[0168] To generate antibodies, an isolated ubiquitin proteasepolypeptide is used as an immunogen to generate antibodies usingstandard techniques for polyclonal and monoclonal antibody preparation.Either the full-length protein or antigenic peptide fragment can beused. Regions having a high antigenicity index are shown in FIG. 3.

[0169] Antibodies are preferably prepared from these regions or fromdiscrete fragments in these regions. However, antibodies can be preparedfrom any region of the peptide as described herein. A preferred fragmentproduces an antibody that diminishes or completely prevents substratehydrolysis or binding. Antibodies can be developed against the entireubiquitin protease or domains of the ubiquitin protease as describedherein. Antibodies can also be developed against specific functionalsites as disclosed herein.

[0170] The antigenic peptide can comprise a contiguous sequence of atleast 12, 14, 15, or 30 amino acid residues. In one embodiment,fragments correspond to regions that are located on the surface of theprotein, e.g., hydrophilic regions. These fragments are not to beconstrued, however, as encompassing any fragments, which may bedisclosed prior to the invention.

[0171] Antibodies can be polyclonal or monoclonal. An intact antibody,or a fragment thereof (e.g. Fab or F(ab′)₂) can be used.

[0172] Detection can be facilitated by coupling (i.e., physicallylinking) the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0173] An appropriate immunogenic preparation can be derived fromnative, recombinantly expressed, or chemically synthesized peptides.

[0174] Antibody Uses

[0175] The antibodies can be used to isolate a ubiquitin protease bystandard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural ubiquitin protease from cells and recombinantly producedubiquitin protease expressed in host cells.

[0176] The antibodies are useful to detect the presence of ubiquitinprotease in cells or tissues to determine the pattern of expression ofthe ubiquitin protease among various tissues in an organism and over thecourse of normal development.

[0177] The antibodies can be used to detect ubiquitin protease in situ,in vitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression.

[0178] The antibodies can be used to assess abnormal tissue distributionor abnormal expression during development.

[0179] Antibody detection of circulating fragments of the full lengthubiquitin protease can be used to identify ubiquitin protease turnover.

[0180] Further, the antibodies can be used to assess ubiquitin proteaseexpression in disease states such as in active stages of the disease orin an individual with a predisposition toward disease related toubiquitin or ubiquitin protease function. When a disorder is caused byan inappropriate tissue distribution, developmental expression, or levelof expression of the ubiquitin protease protein, the antibody can beprepared against the normal ubiquitin protease protein. If a disorder ischaracterized by a specific mutation in the ubiquitin protease,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant ubiquitin protease. However,intracellularly-made antibodies (“intrabodies”) are also encompassed,which would recognize intracellular ubiquitin protease peptide regions.

[0181] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Antibodies can be developed against the whole ubiquitin protease orportions of the ubiquitin protease.

[0182] The diagnostic uses can be applied, not only in genetic testing,but also in monitoring a treatment modality. Accordingly, wheretreatment is ultimately aimed at correcting ubiquitin proteaseexpression level or the presence of aberrant ubiquitin proteases andaberrant tissue distribution or developmental expression, antibodiesdirected against the ubiquitin protease or relevant fragments can beused to monitor therapeutic efficacy.

[0183] Antibodies accordingly can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.

[0184] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic ubiquitin protease can beused to identify individuals that require modified treatment modalities.

[0185] The antibodies are also useful as diagnostic tools as animmunological marker for aberrant ubiquitin protease analyzed byelectrophoretic mobility, isoelectric point, tryptic peptide digest, andother physical assays known to those in the art.

[0186] The antibodies are also useful for tissue typing. Thus, where aspecific ubiquitin protease has been correlated with expression in aspecific tissue, antibodies that are specific for this ubiquitinprotease can be used to identify a tissue type.

[0187] The antibodies are also useful in forensic identification.Accordingly, where an individual has been correlated with a specificgenetic polymorphism resulting in a specific polymorphic protein, anantibody specific for the polymorphic protein can be used as an aid inidentification.

[0188] The antibodies are also useful for inhibiting ubiquitin proteasefunction, for example, blocking ubiquitin or polyubiquitin binding, orbinding to ubiquitinated substrate or substrate remnants.

[0189] These uses can also be applied in a therapeutic context in whichtreatment involves inhibiting ubiquitin protease function. An antibodycan be used, for example, to block ubiquitin binding. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact ubiquitin protease associated with a cell.

[0190] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. For an overview of thistechnology for producing human antibodies, see Lonberg et al. (1995)Int. Rev. Immunol. 13:65-93. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, e.g., U.S. Pat.No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S.Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806.

[0191] The invention also encompasses kits for using antibodies todetect the presence of a ubiquitin protease protein in a biologicalsample. The kit can comprise antibodies such as a labeled or labelableantibody and a compound or agent for detecting ubiquitin protease in abiological sample; means for determining the amount of ubiquitinprotease in the sample; and means for comparing the amount of ubiquitinprotease in the sample with a standard.

[0192] The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectubiquitin protease.

[0193] Polynucleotides

[0194] The nucleotide sequence in SEQ ID NO:2 was obtained by sequencingthe deposited human cDNA. Accordingly, the sequence of the depositedclone is controlling as to any discrepancies between the two and anyreference to the sequence of SEQ ID NO:2 includes reference to thesequence of the deposited cDNA.

[0195] The specifically disclosed cDNA comprises the coding region and5′ and 3′ untranslated sequences in SEQ ID NO:2.

[0196] The invention provides isolated polynucleotides encoding thenovel ubiquitin protease. The term “ubiquitin protease polynucleotide”or “ubiquitin protease nucleic acid” refers to the sequence shown in SEQID NO:2 or in the deposited cDNA. The term “ubiquitin proteasepolynucleotide” or “ubiquitin protease nucleic acid” further includesvariants and fragments of the ubiquitin protease polynucleotide.

[0197] An “isolated” ubiquitin protease nucleic acid is one that isseparated from other nucleic acid present in the natural source of theubiquitin protease nucleic acid. Preferably, an “isolated” nucleic acidis free of sequences which naturally flank the ubiquitin proteasenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. However, there can be some flanking nucleotidesequences, for example up to about 5 KB. The important point is that theubiquitin protease nucleic acid is isolated from flanking sequences suchthat it can be subjected to the specific manipulations described herein,such as recombinant expression, preparation of probes and primers, andother uses specific to the ubiquitin protease nucleic acid sequences.

[0198] Moreover, an “isolated” nucleic acid molecule, such as a cDNA orRNA molecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

[0199] In some instances, the isolated material will form part of acomposition (for example, a crude extract containing other substances),buffer system or reagent mix. In other circumstances, the material maybe purified to essential homogeneity, for example as determined by PAGEor column chromatography such as HPLC. Preferably, an isolated nucleicacid comprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

[0200] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0201] In some instances, the isolated material will form part of acomposition (or example, a crude extract containing other substances),buffer system or reagent mix. In other circumstances, the material maybe purified to essential homogeneity, for example as determined by PAGEor column chromatography such as HPLC. Preferably, an isolated nucleicacid comprises at least about 50, 80 or 90% (on a molar basis) of allmacromolecular species present.

[0202] The ubiquitin protease polynucleotides can encode the matureprotein plus additional amino or carboxyterminal amino acids, or aminoacids interior to the mature polypeptide (when the mature form has morethan one polypeptide chain, for instance). Such sequences may play arole in processing of a protein from precursor to a mature form,facilitate protein trafficking, prolong or shorten protein half-life orfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in situ, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

[0203] The ubiquitin protease polynucleotides include, but are notlimited to, the sequence encoding the mature polypeptide alone, thesequence encoding the mature polypeptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature polypeptide,with or without the additional coding sequences, plus additionalnon-coding sequences, for example introns and non-coding 5′ and 3′sequences such as transcribed but non-translated sequences that play arole in transcription, mRNA processing (including splicing andpolyadenylation signals), ribosome binding and stability of mRNA. Inaddition, the polynucleotide may be fused to a marker sequence encoding,for example, a peptide that facilitates purification.

[0204] Ubiquitin protease polynucleotides can be in the form of RNA,such as mRNA, or in the form DNA, including cDNA and genomic DNAobtained by cloning or produced by chemical synthetic techniques or by acombination thereof. The nucleic acid, especially DNA, can bedouble-stranded or single-stranded. Single-stranded nucleic acid can bethe coding strand (sense strand) or the non-coding strand (anti-sensestrand).

[0205] Ubiquitin protease nucleic acid can comprise the nucleotidesequence shown in SEQ ID NO:2, corresponding to human cDNA.

[0206] In one embodiment, the ubiquitin protease nucleic acid comprisesonly the coding region.

[0207] The invention further provides variant ubiquitin proteasepolynucleotides, and fragments thereof, that differ from the nucleotidesequence shown in SEQ ID NO:2 due to degeneracy of the genetic code andthus encode the same protein as that encoded by the nucleotide sequenceshown in SEQ ID NO:2.

[0208] The invention also provides ubiquitin protease nucleic acidmolecules encoding the variant polypeptides described herein. Suchpolynucleotides may be naturally occurring, such as allelic variants(same locus), homologs (different locus), and orthologs (differentorganism), or may be constructed by recombinant DNA methods or bychemical synthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to polynucleotides,cells, or organisms. Accordingly, as discussed above, the variants cancontain nucleotide substitutions, deletions, inversions and insertions.

[0209] Typically, variants have a substantial identity with a nucleicacid molecule of SEQ ID NO:2 and the complements thereof. Variation canoccur in either or both the coding and non-coding regions. Thevariations can produce both conservative and non-conservative amino acidsubstitutions.

[0210] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. These variants comprise a nucleotidesequence encoding a ubiquitin protease that is at least about 60-65%,65-70%, typically at least about 70-75%, more typically at least about80-85%, and most typically at least about 90-95% or more homologous tothe nucleotide sequence shown in SEQ ID NO:2. Such nucleic acidmolecules can readily be identified as being able to hybridize understringent conditions, to the nucleotide sequence shown in SEQ ID NO:2 ora fragment of the sequence. It is understood that stringenthybridization does not indicate substantial homology where it is due togeneral homology, such as poly A sequences, or sequences common to allor most proteins or all deubiquitinating enzymes. Moreover, it isunderstood that variants do not include any of the nucleic acidsequences that may have been disclosed prior to the invention.

[0211] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a polypeptide at least about 60-65%homologous to each other typically remain hybridized to each other. Theconditions can be such that sequences at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 90%, atleast about 95% or more identical to each other remain hybridized to oneanother. Such stringent conditions are known to those skilled in the artand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference. One exampleof stringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2× SSC, 0.1% SDS at 50-65° C. In another non-limitingexample, nucleic acid molecules are allowed to hybridize in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morelow stringency washes in 0.2× SSC/0.1% SDS at room temperature, or byone or more moderate stringency washes in 0.2× SSC/0.1% SDS at 42° C.,or washed in 0.2× SSC/0.1% SDS at 65° C. for high stringency. In oneembodiment, an isolated nucleic acid molecule that hybridizes understringent conditions to the sequence of SEQ ID NO:1 corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

[0212] As understood by those of ordinary skill, the exact conditionscan be determined empirically and depend on ionic strength, temperatureand the concentration of destabilizing agents such as formamide ordenaturing agents such as SDS. Other factors considered in determiningthe desired hybridization conditions include the length of the nucleicacid sequences, base composition, percent mismatch between thehybridizing sequences and the frequency of occurrence of subsets of thesequences within other non-identical sequences. Thus, equivalentconditions can be determined by varying one or more of these parameterswhile maintaining a similar degree of identity or similarity between thetwo nucleic acid molecules.

[0213] The present invention also provides isolated nucleic acids thatcontain a single or double stranded fragment or portion that hybridizesunder stringent conditions to the nucleotide sequence of SEQ ID NO:2 orthe complement of SEQ ID NO:2. In one embodiment, the nucleic acidconsists of a portion of the nucleotide sequence of SEQ ID NO:2 or thecomplement of SEQ ID NO:2. The nucleic acid fragments of the inventionare at least about 15, preferably at least about 18, 20, 23 or 25nucleotides, and can be 30, 40, 50, 100, 200, 500 or more nucleotides inlength. Longer fragments, for example, 30 or more nucleotides in length,which encode antigenic proteins or polypeptides described herein areuseful.

[0214] Furthermore, the invention provides polynucleotides that comprisea fragment of the full-length ubiquitin protease polynucleotides. Thefragment can be single or double-stranded and can comprise DNA or RNA.The fragment can be derived from either the coding or the non-codingsequence.

[0215] In another embodiment an isolated ubiquitin protease nucleic acidencodes the entire coding region. In another embodiment the isolatedubiquitin protease nucleic acid encodes a sequence corresponding to themature protein that may be from about amino acid 6 to the last aminoacid. Other fragments include nucleotide sequences encoding the aminoacid fragments described herein.

[0216] Thus, ubiquitin protease nucleic acid fragments further includesequences corresponding to the domains described herein, subregions alsodescribed, and specific functional sites. Ubiquitin protease nucleicacid fragments also include combinations of the domains, segments, andother functional sites described above. A person of ordinary skill inthe art would be aware of the many permutations that are possible.

[0217] Where the location of the domains or sites have been predicted bycomputer analysis, one of ordinary sill would appreciate that the aminoacid residues constituting these domains can vary depending on thecriteria used to define the domains.

[0218] However, it is understood that a ubiquitin protease fragmentincludes any nucleic acid sequence that does not include the entiregene.

[0219] The invention also provides ubiquitin protease nucleic acidfragments that encode epitope bearing regions of the ubiquitin proteaseproteins described herein.

[0220] Nucleic acid fragments, according to the present invention, arenot to be construed as encompassing those fragments that may have beendisclosed prior to the invention.

[0221] Polynucleotide Uses

[0222] The nucleotide sequences of the present invention can be used asa “query sequence” to perform a search against public databases, forexample, to identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0223] The nucleic acid fragments of the invention provide probes orprimers in assays such as those described below. “Probes” areoligonucleotides that hybridize in a base-specific manner to acomplementary strand of nucleic acid. Such probes include polypeptidenucleic acids, as described in Nielsen et al. (1991) Science254:1497-1500. Typically, a probe comprises a region of nucleotidesequence that hybridizes under highly stringent conditions to at leastabout 15, typically about 20-25, and more typically about 40, 50 or 75consecutive nucleotides of the nucleic acid sequence shown in SEQ IDNO:2 and the complements thereof. More typically, the probe furthercomprises a label, e.g., radioisotope, fluorescent compound, enzyme, orenzyme co-factor.

[0224] As used herein, the term “primer” refers to a single-strandedoligonucleotide which acts as a point of initiation of template-directedDNA synthesis using well-known methods (e.g., PCR, LCR) including, butnot limited to those described herein. The appropriate length of theprimer depends on the particular use, but typically ranges from about 15to 30 nucleotides. The term “primer site” refers to the area of thetarget DNA to which a primer hybridizes. The term “primer pair” refersto a set of primers including a 5′ (upstream) primer that hybridizeswith the 5′ end of the nucleic acid sequence to be amplified and a 3′(downstream) primer that hybridizes with the complement of the sequenceto be amplified.

[0225] The ubiquitin protease polynucleotides are thus useful forprobes, primers, and in biological assays.

[0226] Where the polynucleotides are used to assess ubiquitin proteaseproperties or functions, such as in the assays described herein, all orless than all of the entire cDNA can be useful. Assays specificallydirected to ubiquitin protease functions, such as assessing agonist orantagonist activity, encompass the use of known fragments. Further,diagnostic methods for assessing ubiquitin protease function can also bepracticed with any fragment, including those fragments that may havebeen known prior to the invention. Similarly, in methods involvingtreatment of ubiquitin protease dysfunction, all fragments areencompassed including those, which may have been known in the art.

[0227] The ubiquitin protease polynucleotides are useful as ahybridization probe for cDNA and genomic DNA to isolate a full-lengthcDNA and genomic clones encoding the polypeptide described in SEQ IDNO:1 and to isolate cDNA and genomic clones that correspond to variantsproducing the same polypeptide shown in SEQ ID NO:1 or the othervariants described herein. Variants can be isolated from the same tissueand organism from which the polypeptides shown in SEQ ID NO:1 wereisolated, different tissues from the same organism, or from differentorganisms. This method is useful for isolating genes and cDNA that aredevelopmentally-controlled and therefore may be expressed in the sametissue or different tissues at different points in the development of anorganism.

[0228] The probe can correspond to any sequence along the entire lengthof the gene encoding the ubiquitin protease. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions.

[0229] The nucleic acid probe can be, for example, the full-length cDNAof SEQ ID NO:2 or a fragment thereof, such as an oligonucleotide of atleast 12, 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to mRNAor DNA.

[0230] Fragments of the polynucleotides described herein are also usefulto synthesize larger fragments or full-length polynucleotides describedherein. For example, a fragment can be hybridized to any portion of anmRNA and a larger or full-length cDNA can be produced.

[0231] The fragments are also useful to synthesize antisense moleculesof desired length and sequence.

[0232] Antisense nucleic acids of the invention can be designed usingthe nucleotide sequence of SEQ ID NO:2, and constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

[0233] Additionally, the nucleic acid molecules of the invention can bemodified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670. PNAs can be further modified, e.g., to enhance theirstability, specificity or cellular uptake, by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. The synthesis of PNA-DNA chimeras can be performed as described inHyrup (1996), supra, Finn et al. (1996) Nucleic Acids Res.24(17):3357-63, Mag et al. (1989) Nucleic Acids Res. 17:5973, andPeterser et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

[0234] The nucleic acid molecules and fragments of the invention canalso include other appended groups such as peptides (e.g., for targetinghost cell ubiquitin proteases in vivo), or agents facilitating transportacross the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl.Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.Sci. USA 84:648-652; PCT Publication No. WO 88/0918) or the blood brainbarrier (see, e.g., PCT Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).

[0235] The ubiquitin protease polynucleotides are also useful as primersfor PCR to amplify any given region of a ubiquitin proteasepolynucleotide.

[0236] The ubiquitin protease polynucleotides are also useful forconstructing recombinant vectors. Such vectors include expressionvectors that express a portion of, or all of, the ubiquitin proteasepolypeptides. Vectors also include insertion vectors, used to integrateinto another polynucleotide sequence, such as into the cellular genome,to alter in situ expression of ubiquitin protease genes and geneproducts. For example, an endogenous ubiquitin protease coding sequencecan be replaced via homologous recombination with all or part of thecoding region containing one or more specifically introduced mutations.

[0237] The ubiquitin protease polynucleotides are also useful forexpressing antigenic portions of the ubiquitin protease proteins.

[0238] The ubiquitin protease polynucleotides are also useful as probesfor determining the chromosomal positions of the ubiquitin proteasepolynucleotides by means of in situ hybridization methods, such as FISH.(For a review of this technique, see Verma et al. (1988) HumanChromosomes: A Manual of Basic Techniques (Pergamon Press, New York),and PCR mapping of somatic cell hybrids. The mapping of the sequences tochromosomes is an important first step in correlating these sequenceswith genes associated with disease.

[0239] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0240] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween a gene and a disease mapped to the same chromosomal region, canthen be identified through linkage analysis (co-inheritance ofphysically adjacent genes), described in, for example, Egeland et al.((1987) Nature 325:783-787).

[0241] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with a specified gene,can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations, that are visible from chromosome spreads,or detectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

[0242] The ubiquitin protease polynucleotide probes are also useful todetermine patterns of the presence of the gene encoding the ubiquitinproteases and their variants with respect to tissue distribution, forexample, whether gene duplication has occurred and whether theduplication occurs in all or only a subset of tissues. The genes can benaturally occurring or can have been introduced into a cell, tissue, ororganism exogenously.

[0243] The ubiquitin protease polynucleotides are also usefid fordesigning ribozymes corresponding to all, or a part, of the mRNAproduced from genes encoding the polynucleotides described herein.

[0244] The ubiquitin protease polynucleotides are also useful forconstructing host cells expressing a part, or all, of the ubiquitinprotease polynucleotides and polypeptides.

[0245] The ubiquitin protease polynucleotides are also useful forconstructing transgenic animals expressing all, or a part, of theubiquitin protease polynucleotides and polypeptides.

[0246] The ubiquitin protease polynucleotides are also useful for makingvectors that express part, or all, of the ubiquitin proteasepolypeptides.

[0247] The ubiquitin protease polynucleotides are also useful ashybridization probes for determining the level of ubiquitin proteasenucleic acid expression. Accordingly, the probes can be used to detectthe presence of, or to determine levels of, ubiquitin protease nucleicacid in cells, tissues, and in organisms. The nucleic acid whose levelis determined can be DNA or RNA. Accordingly, probes corresponding tothe polypeptides described herein can be used to assess gene copy numberin a given cell, tissue, or organism. This is particularly relevant incases in which there has been an amplification of the ubiquitin proteasegenes.

[0248] Alternatively, the probe can be used in an in situ hybridizationcontext to assess the position of extra copies of the ubiquitin proteasegenes, as on extrachromosomal elements or as integrated into chromosomesin which the ubiquitin protease gene is not normally found, for exampleas a homogeneously staining region.

[0249] These uses are relevant for diagnosis of disorders involving anincrease or decrease in ubiquitin protease expression relative tonormal, such as a proliferative disorder, a differentiative ordevelopmental disorder, or a hematopoietic disorder.

[0250] The ubiquitin protease is expressed in tissues including, but notlimited to normal human thymus, testes, brain, breast, ovary, skeletalmuscle, liver, prostate, and thyroid. As such, the gene is particularlyrelevant for the treatment of disorders involving these tissues. Thegene is also expressed in fetal kidney, fetal heart, and fetal liver.The gene is also expressed in liver metastases derived from colon, andmalignant lung and breast and therefore, treatment is relevant to thesedisorders.

[0251] Disorders involving the above tissues are discussed herein above.

[0252] Thus, the present invention provides a method for identifying adisease or disorder associated with aberrant expression or activity ofubiquitin protease nucleic acid, in which a test sample is obtained froma subject and nucleic acid (e.g., mRNA, genomic DNA) is detected,wherein the presence of the nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant expression or activity of the nucleic acid.

[0253] One aspect of the invention relates to diagnostic assays fordetermining nucleic acid expression as well as activity in the contextof a biological sample (e.g., blood, serum, cells, tissue) to determinewhether an individual has a disease or disorder, or is at risk ofdeveloping a disease or disorder, associated with aberrant nucleic acidexpression or activity. Such assays can be used for prognostic orpredictive purpose to thereby prophylactically treat an individual priorto the onset of a disorder characterized by or associated withexpression or activity of the nucleic acid molecules.

[0254] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0255] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express the ubiquitin protease, suchas by measuring the level of a ubiquitin protease-encoding nucleic acidin a sample of cells from a subject e.g., mRNA or genomic DNA, ordetermining if the ubiquitin protease gene has been mutated.

[0256] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate ubiquitin protease nucleic acidexpression (e.g., antisense, polypeptides, peptidomimetics, smallmolecules or other drugs). A cell is contacted with a candidate compoundand the expression of mRNA determined. The level of expression of themRNA in the presence of the candidate compound is compared to the levelof expression of the mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Themodulator can bind to the nucleic acid or indirectly modulateexpression, such as by interacting with other cellular components thataffect nucleic acid expression.

[0257] Modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g., byadministering the gent to a subject) in patients or in transgenicanimals.

[0258] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the ubiquitin protease gene. The method typically includesassaying the ability of the compound to modulate the expression of theubiquitin protease nucleic acid and thus identifying a compound that canbe used to treat a disorder characterized by undesired ubiquitinprotease nucleic acid expression.

[0259] The assays can be performed in cell-based and cell-free systems.Cell-based assays include cells naturally expressing the ubiquitinprotease nucleic acid or recombinant cells genetically engineered toexpress specific nucleic acid sequences.

[0260] Alternatively, candidate compounds can be assayed in vivo inpatients or in transgenic animals.

[0261] The assay for ubiquitin protease nucleic acid expression caninvolve direct assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the pathway (such as free ubiquitinpool or protein turnover). Further, the expression of genes that are up-or down-regulated in response to the ubiquitin protease activity canalso be assayed. In this embodiment the regulatory regions of thesegenes can be operably linked to a reporter gene such as luciferase.

[0262] Thus, modulators of ubiquitin protease gene expression can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof ubiquitin protease mRNA in the presence of the candidate compound iscompared to the level of expression of ubiquitin protease mRNA in theabsence of the candidate compound. The candidate compound can then beidentified as a modulator of nucleic acid expression based on thiscomparison and be used, for example to treat a disorder characterized byaberrant nucleic acid expression. When expression of mRNA isstatistically significantly greater in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of nucleic acid expression. When nucleic acid expression isstatistically significantly less in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of nucleic acid expression.

[0263] Accordingly, the invention provides methods of treatment, withthe nucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate ubiquitin protease nucleicacid expression. Modulation includes both up-regulation (i.e. activationor agonization) or down-regulation (suppression or antagonization) oreffects on nucleic acid activity (e.g. when nucleic acid is mutated orimproperly modified). Treatment is of disorders characterized byaberrant expression or activity of the nucleic acid, including thedisorders described herein.

[0264] Alternatively, a modulator for ubiquitin protease nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the ubiquitin protease nucleic acid expression.

[0265] The ubiquitin protease polynucleotides are also useful formonitoring the effectiveness of modulating compounds on the expressionor activity of the ubiquitin protease gene in clinical trials or in atreatment regimen. Thus, the gene expression pattern can serve as abarometer for the continuing effectiveness of treatment with thecompound, particularly with compounds to which a patient can developresistance. The gene expression pattern can also serve as a markerindicative of a physiological response of the affected cells to thecompound. Accordingly, such monitoring would allow either increasedadministration of the compound or the administration of alternativecompounds to which the patient has not become resistant. Similarly, ifthe level of nucleic acid expression falls below a desirable level,administration of the compound could be commensurately decreased.

[0266] Monitoring can be, for example, as follows: (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a specified mRNA orgenomic DNA of the invention in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the mRNA or genomic DNAin the post-administration samples; (v) comparing the level ofexpression or activity of the mRNA or genomic DNA in thepre-administration sample with the mRNA or genomic DNA in thepost-administration sample or samples; and (vi) increasing or decreasingthe administration of the agent to the subject accordingly.

[0267] The ubiquitin protease polynucleotides are also useful indiagnostic assays for qualitative changes in ubiquitin protease nucleicacid, and particularly in qualitative changes that lead to pathology.The polynucleotides can be used to detect mutations in ubiquitinprotease genes and gene expression products such as mRNA. Thepolynucleotides can be used as hybridization probes to detectnaturally-occurring genetic mutations in the ubiquitin protease gene andthereby to determine whether a subject with the mutation is at risk fora disorder caused by the mutation. Mutations include deletion, addition,or substitution of one or more nucleotides in the gene, chromosomalrearrangement, such as inversion or transposition, modification ofgenomic DNA, such as aberrant methylation patterns or changes in genecopy number, such as amplification. Detection of a mutated form of theubiquitin protease gene associated with a dysfunction provides adiagnostic tool for an active disease or susceptibility to disease whenthe disease results from overexpression, underexpression, or alteredexpression of a ubiquitin protease.

[0268] Mutations in the ubiquitin protease gene can be detected at thenucleic acid level by a variety of techniques. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way.

[0269] In certain embodiments, detection of the mutation involves theuse of a probe/primer in a polymerase chain reaction (PCR) (see, e.g.U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) PNAS 91:360-364), the latter of which can be particularly usefulfor detecting point mutations in the gene (see Abravaya et al. (1995)Nucleic Acids Res. 23:675-682). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0270] It is anticipated that PCR and/or LCR may be desirable to use asa preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

[0271] Alternative amplification methods include: self sustainedsequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well-known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

[0272] Alternatively, mutations in a ubiquitin protease gene can bedirectly identified, for example, by alterations in restriction enzymedigestion patterns determined by gel electrophoresis.

[0273] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

[0274] Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature.

[0275] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method.

[0276] Furthermore, sequence differences between a mutant ubiquitinprotease gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

[0277] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985) Science230:1242); Cotton et al. (1988) PNAS 85:4397; Saleeba et al. (1992)Meth. Enzymol. 217:286-295), electrophoretic mobility of mutant and wildtype nucleic acid is compared (Orita et al. (1989) PNAS 86:2766; Cottonet al. (1993) Mutat. Res. 285:125-144; and Hayashi et al. (1992) Genet.Anal. Tech. Appl. 9:73-79), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.(1985) Nature 313:495). The sensitivity of the assay may be enhanced byusing RNA (rather than DNA), in which the secondary structure is moresensitive to a change in sequence. In one embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet. 7:5). Examples of other techniques fordetecting point mutations include, selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0278] In other embodiments, genetic mutations can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotideprobes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al.(1996) Nature Medicine 2:753-759). For example, genetic mutations can beidentified in two dimensional arrays containing light-generated DNAprobes as described in Cronin et al. supra. Briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

[0279] The ubiquitin protease polynucleotides are also useful fortesting an individual for a genotype that while not necessarily causingthe disease, nevertheless affects the treatment modality. Thus, thepolynucleotides can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). In the present case, forexample, a mutation in the ubiquitin protease gene that results inaltered affinity for ubiquitin could result in an excessive or decreaseddrug effect with standard concentrations of ubiquitin or analog.Accordingly, the ubiquitin protease polynucleotides described herein canbe used to assess the mutation content of the gene in an individual inorder to select an appropriate compound or dosage regimen for treatment.

[0280] Thus polynucleotides displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0281] The methods can involve obtaining a control biological samplefrom a control subject, contacting the control sample with a compound oragent capable of detecting mRNA, or genomic DNA, such that the presenceof mRNA or genomic DNA is detected in the biological sample, andcomparing the presence of mRNA or genomic DNA in the control sample withthe presence of mRNA or genomic DNA in the test sample.

[0282] The ubiquitin protease polynucleotides are also useful forchromosome identification when the sequence is identified with anindividual chromosome and to a particular location on the chromosome.First, the DNA sequence is matched to the chromosome by in situ or otherchromosome-specific hybridization. Sequences can also be correlated tospecific chromosomes by preparing PCR primers that can be used for PCRscreening of somatic cell hybrids containing individual chromosomes fromthe desired species. Only hybrids containing the chromosome containingthe gene homologous to the primer will yield an amplified fragment.Sublocalization can be achieved using chromosomal fragments. Otherstrategies include prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to chromosome-specific libraries. Furthermapping strategies include fluorescence in situ hybridization, whichallows hybridization with probes shorter than those traditionally used.Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on the chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0283] The ubiquitin protease polynucleotides can also be used toidentify individuals from small biological samples. This can be done forexample using restriction fragment-length polymorphism (RFLP) toidentify an individual. Thus, the polynucleotides described herein areuseful as DNA markers for RFLP (See U.S. Pat. No. 5,272,057).

[0284] Furthermore, the ubiquitin protease sequence can be used toprovide an alternative technique, which determines the actual DNAsequence of selected fragments in the genome of an individual. Thus, theubiquitin protease sequences described herein can be used to prepare twoPCR primers from the 5′ and 3′ ends of the sequences. These primers canthen be used to amplify DNA from an individual for subsequentsequencing.

[0285] Panels of corresponding DNA sequences from individuals preparedin this manner can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences. It is estimatedthat allelic variation in humans occurs with a frequency of about onceper each 500 bases. Allelic variation occurs to some degree in thecoding regions of these sequences, and to a greater degree in thenoncoding regions. The ubiquitin protease sequences can be used toobtain such identification sequences from individuals and from tissue.The sequences represent unique fragments of the human genome. Each ofthe sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes.

[0286] If a panel of reagents from the sequences is used to generate aunique identification database for an individual, those same reagentscan later be used to identify tissue from that individual. Using theunique identification database, positive identification of theindividual, living or dead, can be made from extremely small tissuesamples.

[0287] The ubiquitin protease polynucleotides can also be used inforensic identification procedures. PCR technology can be used toamplify DNA sequences taken from very small biological samples, such asa single hair follicle, body fluids (e.g. blood, saliva, or semen). Theamplified sequence can then be compared to a standard allowingidentification of the origin of the sample.

[0288] The ubiquitin protease polynucleotides can thus be used toprovide polynucleotide reagents, e.g., PCR primers, targeted to specificloci in the human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As described above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to the noncoding region are particularly useful since greaterpolymorphism occurs in the noncoding regions, making it easier todifferentiate individuals using this technique.

[0289] The ubiquitin protease polynucleotides can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue. This is useful in cases in which a forensicpathologist is presented with a tissue of unknown origin. Panels ofubiquitin protease probes can be used to identify tissue by speciesand/or by organ type.

[0290] In a similar fashion, these primers and probes can be used toscreen tissue culture for contamination (i.e. screen for the presence ofa mixture of different types of cells in a culture).

[0291] Alternatively, the ubiquitin protease polynucleotides can be useddirectly to block transcription or translation of ubiquitin proteasegene sequences by means of antisense or ribozyme constructs. Thus, in adisorder characterized by abnormally high or undesirable ubiquitinprotease gene expression, nucleic acids can be directly used fortreatment.

[0292] The ubiquitin protease polynucleotides are thus useful asantisense constructs to control ubiquitin protease gene expression incells, tissues, and organisms. A DNA antisense polynucleotide isdesigned to be complementary to a region of the gene involved intranscription, preventing transcription and hence production ofubiquitin protease protein. An antisense RNA or DNA polynucleotide wouldhybridize to the mRNA and thus block translation of mRNA into ubiquitinprotease protein.

[0293] Examples of antisense molecules useful to inhibit nucleic acidexpression include antisense molecules complementary to a fragment ofthe 5′ untranslated region of SEQ ID NO:2 which also includes the startcodon and antisense molecules which are complementary to a fragment ofthe 3′ untranslated region of SEQ ID NO:2.

[0294] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of ubiquitin proteasenucleic acid. Accordingly, these molecules can treat a disordercharacterized by abnormal or undesired ubiquitin protease nucleic acidexpression. This technique involves cleavage by means of ribozymescontaining nucleotide sequences complementary to one or more regions inthe mRNA that attenuate the ability of the mRNA to be translated.Possible regions include coding regions and particularly coding regionscorresponding to the catalytic and other functional activities of theubiquitin protease protein.

[0295] The ubiquitin protease polynucleotides also provide vectors forgene therapy in patients containing cells that are aberrant in ubiquitinprotease gene expression. Thus, recombinant cells, which include thepatient's cells that have been engineered ex vivo and returned to thepatient, are introduced into an individual where the cells produce thedesired ubiquitin protease protein to treat the individual.

[0296] The invention also encompasses kits for detecting the presence ofa ubiquitin protease nucleic acid in a biological sample. For example,the kit can comprise reagents such as a labeled or labelable nucleicacid or agent capable of detecting ubiquitin protease nucleic acid in abiological sample; means for determining the amount of ubiquitinprotease nucleic acid in the sample; and means for comparing the amountof ubiquitin protease nucleic acid in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect ubiquitinprotease mRNA or DNA.

[0297] Computer Readable Means

[0298] The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of mediums to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequence of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORFs)) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exists in nature or in purified form.

[0299] In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein, “computer readable media” refers to any mediumthat can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Theskilled artisan will readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a nucleotide or aminoacid sequence of the present invention.

[0300] As used herein, “recorded” refers to a process for storinginformation on computer readable medium. The skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide or amino acid sequence information of the present invention.

[0301] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereona nucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number ofdataprocessor structuring formats (e.g., text file or database) in orderto obtain computer readable medium having recorded thereon thenucleotide sequence information of the present invention.

[0302] By providing the nucleotide or amino acid sequences of theinvention in computer readable form, the skilled artisan can routinelyaccess the sequence information for a variety of purposes. For example,one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

[0303] As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized thatcommercially important fragments, such as sequence fragments involved ingene expression and protein processing, may be of shorter length.

[0304] As used herein, “a target structural motif,” or “target motif,”refers to any rationally selected sequence or combination of sequencesin which the sequence(s) are chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Protein targetmotifs include, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

[0305] Computer software is publicly available which allows a skilledartisan to access sequence information provided in a computer readablemedium for analysis and comparison to other sequences. A variety ofknown algorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware includes, but is not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBIA).

[0306] For example, software which implements the BLAST (Altschul et al.(1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp.Chem. 17:203-207) search algorithms on a Sybase system can be used toidentify open reading frames (ORFs) of the sequences of the inventionwhich contain homology to ORFs or proteins from other libraries. SuchORFs are protein encoding fragments and are useful in producingcommercially important proteins such as enzymes used in variousreactions and in the production of commercially useful metabolites.

[0307] Vectors/Host Cells

[0308] The invention also provides vectors containing the ubiquitinprotease polynucleotides. The term “vector” refers to a vehicle,preferably a nucleic acid molecule that can transport the ubiquitinprotease polynucleotides. When the vector is a nucleic acid molecule,the ubiquitin protease polynucleotides are covalently linked to thevector nucleic acid. With this aspect of the invention, the vectorincludes a plasmid, single or double stranded phage, a single or doublestranded RNA or DNA viral vector, or artificial chromosome, such as aBAC, PAC, YAC, OR MAC.

[0309] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the ubiquitin protease polynucleotides. Alternatively, thevector may integrate into the host cell genome and produce additionalcopies of the ubiquitin protease polynucleotides when the host cellreplicates.

[0310] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the ubiquitinprotease polynucleotides. The vectors can function in procaryotic oreukaryotic cells or in both (shuttle vectors).

[0311] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the ubiquitin protease polynucleotidessuch that transcription of the polynucleotides is allowed in a hostcell. The polynucleotides can be introduced into the host cell with aseparate polynucleotide capable of affecting transcription. Thus, thesecond polynucleotide may provide a trans-acting factor interacting withthe cis-regulatory control region to allow transcription of theubiquitin protease polynucleotides from the vector. Alternatively, atrans-acting factor may be supplied by the host cell. Finally, atrans-acting factor can be produced from the vector itself.

[0312] It is understood, however, that in some embodiments,transcription and/or translation of the ubiquitin proteasepolynucleotides can occur in a cell-free system.

[0313] The regulatory sequence to which the polynucleotides describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

[0314] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0315] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

[0316] A variety of expression vectors can be used to express aubiquitin protease polynucleotide. Such vectors include chromosomal,episomal, and virus-derived vectors, for example vectors derived frombacterial plasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0317] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0318] The ubiquitin protease polynucleotides can be inserted into thevector nucleic acid by well-known methodology. Generally, the DNAsequence that will ultimately be expressed is joined to an expressionvector by cleaving the DNA sequence and the expression vector with oneor more restriction enzymes and then ligating the fragments together.Procedures for restriction enzyme digestion and ligation are well knownto those of ordinary skill in the art.

[0319] The vector containing the appropriate polynucleotide can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0320] As described herein, it may be desirable to express thepolypeptide as a fusion protein. Accordingly, the invention providesfusion vectors that allow for the production of the ubiquitin proteasepolypeptides. Fusion vectors can increase the expression of arecombinant protein, increase the solubility of the recombinant protein,and aid in the purification of the protein by acting for example as aligand for affinity purification. A proteolytic cleavage site may beintroduced at the junction of the fusion moiety so that the desiredpolypeptide can ultimately be separated from the fusion moiety.Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al. (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal. (1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) GeneExpression Technology: Methods in Enzymology 185:60-89).

[0321] Recombinant protein expression can be maximized in a hostbacteria by providing a genetic background wherein the host cell has animpaired capacity to proteolytically cleave the recombinant protein.(Gottesman, S. (1990) Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. 119-128). Alternatively, thesequence of the polynucleotide of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al. (1992) Nucleic Acids Res. 20:2111 -2118).

[0322] The ubiquitin protease polynucleotides can also be expressed byexpression vectors that are operative in yeast. Examples of vectors forexpression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234 ), pMFa (Kurjan et al. (1982) Cell 30:933-943),pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0323] The ubiquitin protease polynucleotides can also be expressed ininsect cells using, for example, baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow et al.(1989) Virology 170:31-39).

[0324] In certain embodiments of the invention, the polynucleotidesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)EMBO J. 6:187-195).

[0325] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the ubiquitin proteasepolynucleotides. The person of ordinary skill in the art would be awareof other vectors suitable for maintenance propagation or expression ofthe polynucleotides described herein. These are found for example inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

[0326] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the polynucleotide sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0327] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0328] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y).

[0329] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the ubiquitin protease polynucleotides can beintroduced either alone or with other polynucleotides that are notrelated to the ubiquitin protease polynucleotides such as thoseproviding trans-acting factors for expression vectors. When more thanone vector is introduced into a cell, the vectors can be introducedindependently, co-introduced or joined to the ubiquitin proteasepolynucleotide vector.

[0330] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0331] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the polynucleotides described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0332] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0333] Where secretion of the polypeptide is desired, appropriatesecretion signals are incorporated into the vector. The signal sequencecan be endogenous to the ubiquitin protease polypeptides or heterologousto these polypeptides.

[0334] Where the polypeptide is not secreted into the medium, theprotein can be isolated from the host cell by standard disruptionprocedures, including freeze thaw, sonication, mechanical disruption,use of lysing agents and the like. The polypeptide can then be recoveredand purified by well-known purification methods including ammoniumsulfate precipitation, acid extraction, anion or cationic exchangechromatography, phosphocellulose chromatography, hydrophobic-interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, or high performance liquid chromatography.

[0335] It is also understood that depending upon the host cell inrecombinant production of the polypeptides described herein, thepolypeptides can have various glycosylation patterns, depending upon thecell, or maybe non-glycosylated as when produced in bacteria. Inaddition, the polypeptides may include an initial modified methionine insome cases as a result of a host-mediated process.

[0336] Uses of Vectors and Host Cells

[0337] It is understood that “host cells” and “recombinant host cells”refer not only to the particular subject cell but also to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0338] The host cells expressing the polypeptides described herein, andparticularly recombinant host cells, have a variety of uses. First, thecells are useful for producing ubiquitin protease proteins orpolypeptides that can be further purified to produce desired amounts ofubiquitin protease protein or fragments. Thus, host cells containingexpression vectors are useful for polypeptide production.

[0339] Host cells are also useful for conducting cell-based assaysinvolving the ubiquitin protease or ubiquitin protease fragments. Thus,a recombinant host cell expressing a native ubiquitin protease is usefulto assay for compounds that stimulate or inhibit ubiquitin proteasefunction. This includes disappearance of substrate (polyubiquitin,ubiquitinated substrate protein, ubiquitinated substrate remnants),appearance of end product (ubiquitin monomers, polyubiquitin hydrolyzedfrom substrate or substrate remnant, free substrate that has beenrescued by hydrolysis of ubiquitin), general or specific proteinturnover, and the various other molecular functions described hereinthat include, but are not limited to, substrate recognition, substratebinding, subunit association, and interaction with other cellularcomponents. Modulation of gene expression can occur at the level oftranscription or translation.

[0340] Host cells are also useful for identifying ubiquitin proteasemutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutantubiquitin protease (for example, stimulating or inhibiting function)which may not be indicated by their effect on the native ubiquitinprotease.

[0341] Recombinant host cells are also useful for expressing thechimeric polypeptides described herein to assess compounds that activateor suppress activation or alter specific function by means of aheterologous domain, segment, site, and the like, as disclosed herein.

[0342] Further, mutant ubiquitin proteases can be designed in which oneor more of the various functions is engineered to be increased ordecreased (e.g., binding to ubiquitin, polyubiquitin, or ubiquitinatedprotein substrate) and used to augment or replace ubiquitin proteaseproteins in an individual. Thus, host cells can provide a therapeuticbenefit by replacing an aberrant ubiquitin protease or providing anaberrant ubiquitin protease that provides a therapeutic result. In oneembodiment, the cells provide ubiquitin proteases that are abnormallyactive.

[0343] In another embodiment, the cells provide ubiquitin proteases thatare abnormally inactive. These ubiquitin proteases can compete withendogenous ubiquitin proteases in the individual.

[0344] In another embodiment, cells expressing ubiquitin proteases thatcannot be activated, are introduced into an individual in order tocompete with endogenous ubiquitin proteases for ubiquitin substrates.For example, in the case in which excessive ubiquitin substrate oranalog is part of a treatment modality, it may be necessary toinactivate this molecule at a specific point in treatment. Providingcells that compete for the molecule , but which cannot be affected byubiquitin protease activation would be beneficial.

[0345] Homologously recombinant host cells can also be produced thatallow the in situ alteration of endogenous 23413 polynucleotidesequences in a host cell genome. The host cell includes, but is notlimited to, a stable cell line, cell in vivo, or cloned microorganism.This technology is more fully described in WO 93/09222, WO 91/12650, WO91/06667, U.S. Pat. No. 5,272,071, and U.S. Pat. No. 5,641,670. Briefly,specific polynucleotide sequences corresponding to the 23413polynucleotides or sequences proximal or distal to a 23413 gene areallowed to integrate into a host cell genome by homologous recombinationwhere expression of the gene can be affected. In one embodiment,regulatory sequences are introduced that either increase or decreaseexpression of an endogenous sequence. Accordingly, a 23413 protein canbe produced in a cell not normally producing it. Alternatively,increased expression of 23413 protein can be effected in a cell normallyproducing the protein at a specific level. Further, expression can bedecreased or eliminated by introducing a specific regulatory sequence.The regulatory sequence can be heterologous to the 23413 proteinsequence or can be a homologous sequence with a desired mutation thataffects expression. Alternatively, the entire gene can be deleted. Theregulatory sequence can be specific to the host cell or capable offunctioning in more than one cell type. Still further, specificmutations can be introduced into any desired region of the gene toproduce mutant 23413 proteins. Such mutations could be introduced, forexample, into the specific functional regions such as the ligand-bindingsite.

[0346] In one embodiment, the host cell can be a fertilized oocyte orembryonic stem cell that can be used to produce a transgenic animalcontaining the altered ubiquitin protease gene. Alternatively, the hostcell can be a stem cell or other early tissue precursor that gives riseto a specific subset of cells and can be used to produce transgenictissues in an animal. See also Thomas et al., Cell 51:503 (1987) for adescription of homologous recombination vectors. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced gene has homologously recombined withthe endogenous ubiquitin protease gene is selected (see e.g., Li, E. etal. (1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos. WO 90/11354; WO 91/01140; and WO93/04169.

[0347] The genetically engineered host cells can be used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a ubiquitinprotease protein and identifying and evaluating modulators of ubiquitinprotease protein activity.

[0348] Other examples of transgenic animals include non-human primates,sheep, dogs, cows, goats, chickens, and amphibians.

[0349] In one embodiment, a host cell is a fertilized oocyte or anembryonic stem cell into which ubiquitin protease polynucleotidesequences have been introduced.

[0350] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the ubiquitin proteasenucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0351] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the ubiquitin protease protein toparticular cells.

[0352] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0353] In another embodiment, transgenic non-human animals can beproduced which contain selected systems, which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. (1991) Science251:1351-1355. If a cre/loxP recombinase system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe Cre recombinase and a selected protein is required. Such animals canbe provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected protein and the other containing a transgeneencoding a recombinase.

[0354] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wihnut et al.(1997) Nature 385:810-813 and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to a pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0355] Transgenic animals containing recombinant cells that express thepolypeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could affect, for example,binding, activation, and protein turnover, may not be evident from invitro cell-free or cell-based assays. Accordingly, it is useful toprovide non-human transgenic animals to assay in vivo ubiquitin proteasefunction, including substrate interaction, the effect of specific mutantubiquitin proteases on ubiquitin protease function and substrateinteraction, and the effect of chimeric ubiquitin proteases. It is alsopossible to assess the effect of null mutations, that is mutations thatsubstantially or completely eliminate one or more ubiquitin proteasefunctions.

[0356] In general, methods for producing transgenic animals includeintroducing a nucleic acid sequence according to the present invention,the nucleic acid sequence capable of expressing the receptor protein ina transgenic animal, into a cell in culture or in vivo. When introducedin vivo, the nucleic acid is introduced into an intact organism suchthat one or more cell types and, accordingly, one or more tissue types,express the nucleic acid encoding the receptor protein. Alternatively,the nucleic acid can be introduced into virtually all cells in anorganism by transfecting a cell in culture, such as an embryonic stemcell, as described herein for the production of transgenic animals, andthis cell can be used to produce an entire transgenic organism. Asdescribed, in a further embodiment, the host cell can be a fertilizedoocyte. Such cells are then allowed to develop in a female foster animalto produce the transgenic organism.

[0357] Pharmaceutical Compositions

[0358] The ubiquitin protease nucleic acid molecules, protein modulatorsof the protein, and antibodies (also referred to herein as “activecompounds”) can be incorporated into pharmaceutical compositionssuitable for administration to a subject, e.g., a human. Suchcompositions typically comprise the nucleic acid molecule, protein,modulator, or antibody and a pharmaceutically acceptable carrier.

[0359] The term “administer” is used in its broadest sense and includesany method of introducing the compositions of the present invention intoa subject. This includes producing polypeptides or polynucleotides invivo as by transcription or translation, in vivo, of polynucleotidesthat have been exogenously introduced into a subject. Thus, polypeptidesor nucleic acids produced in the subject from the exogenous compositionsare encompassed in the term “administer.”

[0360] As used herein the language “pharmaceutically acceptable carrier”is intended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, such media can be used in thecompositions of the invention. Supplementary active compounds can alsobe incorporated into the compositions. A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

[0361] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0362] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a ubiquitin protease protein or anti-ubiquitinprotease antibody) in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0363] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For oral administration, the agent can be contained in entericforms to survive the stomach or further coated or mixed to be releasedin a particular region of the GI tract by known methods. For the purposeof oral therapeutic administration, the active compound can beincorporated with excipients and used in the form of tablets, troches,or capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0364] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser,which contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

[0365] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0366] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0367] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0368] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. “Dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0369] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see e.g., Chen et al. (1994) PNAS 91:3054-3057). The pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g. retroviral vectors, the pharmaceutical preparation caninclude one or more cells which produce the gene delivery system.

[0370] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0371] As defined herein, a therapeutically effective amount of proteinor polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight.

[0372] The skilled artisan will appreciate that certain factors mayinfluence the dosage required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a protein, polypeptide, or antibodycan include a single treatment or, preferably, can include a series oftreatments. In a preferred example, a subject is treated with antibody,protein, or polypeptide in the range of between about 0.1 to 20 mg/kgbody weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody, protein, orpolypeptide used for treatment may increase or decrease over the courseof a particular treatment. Changes in dosage may result and becomeapparent from the results of diagnostic assays as described herein.

[0373] The present invention encompasses agents which modulateexpression or activity. An agent may, for example, be a small molecule.For example, such small molecules include, but are not limited to,peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

[0374] It is understood that appropriate doses of small molecule agentsdepends upon a number of factors within the ken of the ordinarilyskilled physician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

[0375] This invention may be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will fully conveythe invention to those skilled in the art. Many modifications and otherembodiments of the invention will come to mind in one skilled in the artto which this invention pertains having the benefit of the teachingspresented in the foregoing description. Although specific terms areemployed, they are used as in the art unless otherwise indicated.

1 12 1 372 PRT Homosapiens 1 Met Ser Lys Ala Phe Gly Leu Leu Arg Gln IleCys Gln Ser Ile Leu 1 5 10 15 Ala Glu Ser Ser Gln Ser Pro Ala Asp LeuGlu Glu Lys Lys Glu Glu 20 25 30 Asp Ser Asn Met Lys Arg Glu Gln Pro ArgGlu Arg Pro Arg Ala Trp 35 40 45 Asp Tyr Pro His Gly Leu Val Gly Leu HisAsn Ile Gly Gln Thr Cys 50 55 60 Cys Leu Asn Ser Leu Ile Gln Val Phe ValMet Asn Val Asp Phe Thr 65 70 75 80 Arg Ile Leu Lys Arg Ile Thr Val ProArg Gly Ala Asp Glu Gln Arg 85 90 95 Arg Ser Val Pro Phe Gln Met Leu LeuLeu Leu Glu Lys Met Gln Asp 100 105 110 Ser Arg Gln Lys Ala Val Arg ProLeu Glu Leu Ala Tyr Cys Leu Gln 115 120 125 Lys Cys Asn Val Pro Leu PheVal Gln His Asp Ala Ala Gln Leu Tyr 130 135 140 Leu Lys Leu Trp Asn LeuIle Lys Asp Gln Ile Thr Asp Val His Leu 145 150 155 160 Val Glu Arg LeuGln Ala Leu Tyr Thr Ile Arg Val Lys Asp Ser Leu 165 170 175 Ile Cys ValAsp Cys Ala Met Glu Ser Ser Arg Asn Ser Ser Met Leu 180 185 190 Thr LeuPro Leu Ser Leu Phe Asp Val Asp Ser Lys Pro Leu Lys Thr 195 200 205 LeuGlu Asp Ala Leu His Cys Phe Phe Gln Pro Arg Glu Leu Ser Ser 210 215 220Lys Ser Lys Cys Phe Cys Glu Asn Cys Gly Lys Lys Thr Arg Gly Lys 225 230235 240 Gln Val Leu Lys Leu Thr His Leu Pro Gln Thr Leu Thr Ile His Leu245 250 255 Met Arg Phe Ser Ile Arg Asn Ser Gln Thr Arg Lys Ile Cys HisSer 260 265 270 Leu Tyr Phe Pro Gln Ser Leu Asp Phe Ser Gln Ile Leu ProMet Lys 275 280 285 Arg Glu Ser Cys Asp Ala Glu Glu Gln Ser Gly Gly GlnTyr Glu Leu 290 295 300 Phe Ala Val Ile Ala His Val Gly Met Ala Asp SerGly His Tyr Cys 305 310 315 320 Val Tyr Ile Arg Asn Ala Val Asp Gly LysTrp Phe Cys Phe Asn Asp 325 330 335 Ser Asn Ile Cys Leu Val Ser Trp GluAsp Ile Gln Cys Thr Tyr Gly 340 345 350 Asn Pro Asn Tyr His Trp Gln GluThr Ala Tyr Leu Leu Val Tyr Met 355 360 365 Lys Met Glu Cys 370 2 1869DNA Homo sapiens misc_feature (0)...(0) 23413 Ubiquitin 2 caccccgcgtccgcagcagc ggaggctgga cgcttgcatg gcgcttgaga gattccatcg 60 tgcctggctcacataagcgc ttcctggaag tgaagtcgtg ctgtcctgaa cgcgggccag 120 gcagctgcggcctgggggtt ttggagtgat cacga atg agc aag gcg ttt ggg 173 Met Ser Lys AlaPhe Gly 1 5 ctc ctg agg caa atc tgt cag tcc atc ctg gct gag tcc tcg cagtcc 221 Leu Leu Arg Gln Ile Cys Gln Ser Ile Leu Ala Glu Ser Ser Gln Ser10 15 20 ccg gca gat ctt gaa gaa aag aag gaa gaa gac agc aac atg aag aga269 Pro Ala Asp Leu Glu Glu Lys Lys Glu Glu Asp Ser Asn Met Lys Arg 2530 35 gag cag ccc aga gag cgt ccc agg gcc tgg gac tac cct cat ggc ctg317 Glu Gln Pro Arg Glu Arg Pro Arg Ala Trp Asp Tyr Pro His Gly Leu 4045 50 gtt ggt tta cac aac att gga cag acc tgc tgc ctt aac tcc ttg att365 Val Gly Leu His Asn Ile Gly Gln Thr Cys Cys Leu Asn Ser Leu Ile 5560 65 70 cag gtg ttc gta atg aat gtg gac ttc acc agg ata ttg aag agg atc413 Gln Val Phe Val Met Asn Val Asp Phe Thr Arg Ile Leu Lys Arg Ile 7580 85 acg gtg ccc agg gga gct gac gag cag agg aga agc gtc cct ttc cag461 Thr Val Pro Arg Gly Ala Asp Glu Gln Arg Arg Ser Val Pro Phe Gln 9095 100 atg ctt ctg ctg ctg gag aag atg cag gac agc cgg cag aaa gca gtg509 Met Leu Leu Leu Leu Glu Lys Met Gln Asp Ser Arg Gln Lys Ala Val 105110 115 cgg ccc ctg gag ctg gcc tac tgc ctg cag aag tgc aac gtg ccc ttg557 Arg Pro Leu Glu Leu Ala Tyr Cys Leu Gln Lys Cys Asn Val Pro Leu 120125 130 ttt gtc caa cat gat gct gcc caa ctg tac ctc aaa ctc tgg aac ctg605 Phe Val Gln His Asp Ala Ala Gln Leu Tyr Leu Lys Leu Trp Asn Leu 135140 145 150 att aag gac cag atc act gat gtg cac ttg gtg gag aga ctg caggcc 653 Ile Lys Asp Gln Ile Thr Asp Val His Leu Val Glu Arg Leu Gln Ala155 160 165 ctg tat acg atc cgg gtg aag gac tcc ttg att tgc gtt gac tgtgcc 701 Leu Tyr Thr Ile Arg Val Lys Asp Ser Leu Ile Cys Val Asp Cys Ala170 175 180 atg gag agt agc aga aac agc agc atg ctc acc ctc cca ctt tctctt 749 Met Glu Ser Ser Arg Asn Ser Ser Met Leu Thr Leu Pro Leu Ser Leu185 190 195 ttt gat gtg gac tca aag ccc ctg aag aca ctg gag gac gcc ctgcac 797 Phe Asp Val Asp Ser Lys Pro Leu Lys Thr Leu Glu Asp Ala Leu His200 205 210 tgc ttc ttc cag ccc agg gag tta tca agc aaa agc aag tgc ttctgt 845 Cys Phe Phe Gln Pro Arg Glu Leu Ser Ser Lys Ser Lys Cys Phe Cys215 220 225 230 gag aac tgt ggg aag aag acc cgt ggg aaa cag gtc ttg aagctg acc 893 Glu Asn Cys Gly Lys Lys Thr Arg Gly Lys Gln Val Leu Lys LeuThr 235 240 245 cat ttg ccc cag acc ctg aca atc cac ctc atg cga ttc tccatc agg 941 His Leu Pro Gln Thr Leu Thr Ile His Leu Met Arg Phe Ser IleArg 250 255 260 aat tca cag acg aga aag atc tgc cac tcc ctg tac ttc ccccag agc 989 Asn Ser Gln Thr Arg Lys Ile Cys His Ser Leu Tyr Phe Pro GlnSer 265 270 275 ttg gat ttc agc cag atc ctt cca atg aag cga gag tct tgtgat gct 1037 Leu Asp Phe Ser Gln Ile Leu Pro Met Lys Arg Glu Ser Cys AspAla 280 285 290 gag gag cag tct gga ggg cag tat gag ctt ttt gct gtg attgcg cac 1085 Glu Glu Gln Ser Gly Gly Gln Tyr Glu Leu Phe Ala Val Ile AlaHis 295 300 305 310 gtg gga atg gca gac tcc ggt cat tac tgt gtc tac atccgg aat gct 1133 Val Gly Met Ala Asp Ser Gly His Tyr Cys Val Tyr Ile ArgAsn Ala 315 320 325 gtg gat gga aaa tgg ttc tgc ttc aat gac tcc aat atttgc ttg gtg 1181 Val Asp Gly Lys Trp Phe Cys Phe Asn Asp Ser Asn Ile CysLeu Val 330 335 340 tcc tgg gaa gac atc cag tgt acc tac gga aat cct aactac cac tgg 1229 Ser Trp Glu Asp Ile Gln Cys Thr Tyr Gly Asn Pro Asn TyrHis Trp 345 350 355 cag gaa act gca tat ctt ctg gtt tac atg aag atg gagtgc 1271 Gln Glu Thr Ala Tyr Leu Leu Val Tyr Met Lys Met Glu Cys 360 365370 taatggaaat gcccaaaacc ttcagagatt gacacgctgt cattttccat ttccgttcct1331 ggatctacgg agtcttctaa gagattttgc aatgaggaga agcattgttt tcaaactata1391 taactgagcc ttatttataa ttagggatat tatcaaaata tgtaaccatg aggcccctca1451 ggtcctgatc agtcagaatg gatgctttca ccagcagacc cggccatgtg gctgctcggt1511 cctgggtgct cgctgctgtg caagacatta gccctttagt tatgagcctg tgggaacttc1571 aggggttccc agtggggaga gcagtggcag tgggaggcat ctgggggcca aaggtcagtg1631 gcagggggta tttcagtatt atacaactgc tgtgaccaga cttgtatact ggctgaatat1691 cagtgctgtt tgtaattttt cactttgaga accaacatta attccatatg aatcaagtgt1751 tttgtaactg ctattcattt attcagcaaa tatttattga tcatctcttc tccataagat1811 agtgtgataa acacagtcat gaataaagtt attttccaca aaaaaaaaaa aaaaaagg1869 3 18 PRT Artificial Sequence UCH-1 consensus domain 3 Gly Leu GluAsn Leu Gly Asn Thr Cys Tyr Met Asn Ser Val Leu Gln 1 5 10 15 Cys Leu 444 PRT Artificial Sequence UCH-2 consensus domain 4 Tyr Asp Leu Tyr GlyVal Val Cys His Tyr Gly Ala Thr Leu Ser Gly 1 5 10 15 Gly His Tyr ThrAla Tyr Val Lys Lys Glu Leu Glu His Glu Val Leu 20 25 30 Lys Asn Lys TrpTyr Leu Phe Asp Asp Glu Thr Val 35 40 5 292 PRT Artificial SequenceProDom consensus sequence 5 Ser Trp Asp Ser Lys Arg Gly Pro Gly Tyr ThrGly Leu Lys Asn Leu 1 5 10 15 Gly Asn Thr Cys Tyr Met Asn Ser Val LeuGln Cys Leu Tyr His Val 20 25 30 Pro Pro Leu Arg Glu Tyr Phe Leu Glu AspGlu Tyr Glu Ser Glu Met 35 40 45 Val Asn Asn Glu Ser Asn Pro Leu Gly MetLys Gly Glu Leu Ala Thr 50 55 60 Ala Tyr Ala Lys Leu Val His Gln Met TrpSer Asn Ser Ser Asn Lys 65 70 75 80 Ser Val Ala Pro Thr Gln Phe Leu ThrThr Val Gly Lys Phe Ser Pro 85 90 95 Gln Phe Ser Glu Gly Tyr Gln Gln GlnAsp Ser Gln Glu Phe Leu Lys 100 105 110 Phe Leu Gln Asp Asp Ala His GluAsp Phe Asn Ser Leu Met Glu Lys 115 120 125 Pro Tyr Val Glu Glu Gln ValLys Asp Ser Asn Glu Lys Ser Thr Ala 130 135 140 Leu Val Asn Val Ser GluGlu Ala Trp Glu Asn His Lys Lys Arg Asn 145 150 155 160 Asp Ser Ile IleThr Asp Ile Phe Gln Gly Gln Phe Lys Ser Thr Ile 165 170 175 Lys Cys ProSer Cys Glu His Thr Ser Glu Thr Thr Phe Glu Pro Phe 180 185 190 Met AspLeu Ser Leu Pro Ile Pro Ser Asp Ser Ala Asp Asn His Gln 195 200 205 AsnLeu Gln Asp Cys Leu Glu Ser Phe Thr Lys Lys Glu Thr Leu Glu 210 215 220Gly Asp Asn Lys Trp Tyr Cys Pro Lys Cys Lys Lys Lys Gln Glu Ala 225 230235 240 Thr Lys Lys Leu Asp Ile Trp Lys Leu Pro Pro Val Leu Val Ile His245 250 255 Leu Lys Arg Phe Ser Tyr Asp Arg Gln Trp Gly Arg Arg Asp LysLeu 260 265 270 Asn Thr Thr Val Glu Phe Pro Leu Glu Asp Leu Asp Met SerPro Tyr 275 280 285 Val Asp Lys Lys 290 6 84 PRT Artificial SequenceProDom consensus sequence 6 Ile Met Ser Glu Ser Thr Ser Ser Asn Glu ThrLys Ser Asn Asn Pro 1 5 10 15 Tyr Lys Tyr Glu Leu Tyr Gly Val Ile ValHis Ser Gly Ser Ser Met 20 25 30 Asn Gly Gly His Tyr Val Ala Tyr Val LysAsn Arg Ser Lys Asn Asn 35 40 45 Gly Lys Trp Tyr Lys Phe Asp Asp Glu LysVal Thr Glu Val Ser Glu 50 55 60 Glu Asp Val Ile Lys Thr Ser Gly Asp SerSer Ala Tyr Ile Leu Phe 65 70 75 80 Tyr Glu Arg Val 7 79 PRT ArtificialSequence ProDom consensus sequence 7 Ser Ile Glu Lys Ser Ile Lys Asp PhePhe Asn Pro Glu Leu Ile Lys 1 5 10 15 Val Asp Lys Glu Gln Lys Gly TyrVal Cys Glu Lys Cys His Lys Thr 20 25 30 Thr Asn Ala Val Lys His Ser SerIle Leu Arg Ala Pro Glu Thr Leu 35 40 45 Leu Val His Leu Lys Lys Phe ArgPhe Asn Gly Thr Ser Ser Ser Lys 50 55 60 Met Lys Gln Ala Val Ser Tyr ProMet Phe Leu Asp Leu Thr Glu 65 70 75 8 87 PRT Artificial Sequence ProDomconsensus sequence 8 Lys Gly Lys Val Ile Lys Lys Asp Val Val Val Gln LeuPro Asp Ile 1 5 10 15 Leu Ile Val His Leu Ser Arg Ser Thr Phe Asn GlyIle Thr Tyr Ser 20 25 30 Arg Asn Pro Cys Asn Val Lys Phe Gly Glu Arg IleThr Leu Ser Glu 35 40 45 Tyr Thr Leu Ala Glu Ser Gly Thr Ile Thr Glu AsnArg Gln Val Lys 50 55 60 Tyr Asn Leu Lys Ser Val Val Lys His Thr Gly SerHis Ser Ser Gly 65 70 75 80 His Tyr Met Cys Tyr Arg Arg 85 9 142 PRTArtificial Sequence ProDom consensus sequence 9 Met His Ala His Pro ProIle Arg Ser Tyr Phe Glu Ile Glu Met Phe 1 5 10 15 Ile Ala Tyr Glu CysLys Ser Cys Lys His Val Ser Asn Ala Pro Asp 20 25 30 Lys Ala Ile Tyr ValSer Ile Asp Leu Ser Ser Lys Thr Lys Gly Thr 35 40 45 Met Gln Ser Met ValAsp Lys Met Ala Asn Pro Ile Pro Val Val Gly 50 55 60 Met Asn Cys Lys SerCys Gly Gln Glu Thr Leu Cys Ser Thr Thr Arg 65 70 75 80 Phe Thr Lys LeuPro Glu Val Leu Leu Tyr Phe Val Pro Arg Val Lys 85 90 95 Asp Gln Gln ArgGly Lys Asp Met Thr Val Leu Asn Val Gln Arg Gln 100 105 110 Leu Ile LeuLys Asp Asp Asn Asn Ala His Asn Tyr Glu Leu Cys Ser 115 120 125 Phe IleAla His Cys Gly Lys Asn Gly Asp Asn Gly His Tyr 130 135 140 10 48 PRTArtificial Sequence ProDom consensus sequence 10 Thr Leu His Leu Thr LeuTyr Asn Pro Ser Asn Arg Pro Leu Thr Ile 1 5 10 15 Arg Arg Gly Asp LeuVal Ala Val Ala Val Pro Cys Tyr Cys His Pro 20 25 30 Ala Lys Ala Pro SerGln Asp Val Cys Phe Cys Glu Glu Arg Gly Arg 35 40 45 11 40 PRTArtificial Sequence ProDom consensus sequence 11 Cys Arg His Ile Gln AspHis Cys Glu Gln Gln Ile Gln Asp Leu Glu 1 5 10 15 Arg Arg His Arg GlnGln Gln Gly His Leu Arg Asp Gln His Gln Glu 20 25 30 Glu Arg Arg Asp TrpGlu Phe Pro 35 40 12 81 PRT Artificial Sequence ProDom consensussequence 12 Asp Ala Ser Lys Gln Gly Tyr Gln His Phe Phe Ala Leu Leu GlyAla 1 5 10 15 Ala Ser Ala Val Thr Thr Gly His Pro Glu Ala Arg Lys LeuLeu Asp 20 25 30 Tyr Thr Ile Glu Ile Ile Glu Lys Tyr Phe Trp Ser Glu GluGlu Gln 35 40 45 Met Cys Leu Glu Ser Trp Asp Glu Ala Phe Ser Lys Thr GluGlu Tyr 50 55 60 Arg Gly Gly Asn Ala Asn Met His Ala Val Glu Ala Phe LeuIle Val 65 70 75 80 Tyr

That which is claimed:
 1. An isolated nucleic acid molecule comprising anucleotide sequence selected from the group consisting of: (a) anucleotide sequence having at least 65% sequence identity to thenucleotide sequence set forth in SEQ ID NO:2, wherein said sequenceidentity is calculated using the GAP algorithm with a gap weight of 40and a length weight of 4 and said nucleotide sequence encodes apolypeptide having ubiquitin protease activity; and (b) a nucleotidesequence complementary the nucleotide sequence of (a).
 2. The nucleicacid molecule of claim 1, wherein said nucleic acid molecule comprises anucleotide sequence selected from the group consisting of: (a) anucleotide sequence having at least 70% sequence identity to thenucleotide sequence set forth in SEQ ID NO:2, wherein said sequenceidentity is calculated using the GAP algorithm with a gap weight of 40and a length weight of 4 and said nucleotide sequence encodes apolypeptide having ubiquitin protease activity; (b) a nucleotidesequence complementary to the nucleotide sequence of (a).
 3. The nucleicacid molecule of claim 2, wherein said nucleic acid molecule comprises anucleotide sequence selected from the group consisting of: (a) anucleotide sequence having at least 80% sequence identity to thenucleotide sequence set forth in SEQ ID NO:2, wherein said sequenceidentity is calculated using the GAP algorithm with a gap weight of 40and a length weight of 4 and said nucleotide sequence encodes apolypeptide having ubiquitin protease activity; (b) a nucleotidesequence complementary to the nucleotide sequence of (a).
 4. The nucleicacid molecule of claim 3, wherein said nucleic acid molecule comprises anucleotide sequence selected from the group consisting of: (a) anucleotide sequence having at least 90% sequence identity to thenucleotide sequence set forth in SEQ ID NO:2, wherein said sequenceidentity is calculated using the GAP algorithm with a gap weight of 40and a length weight of 4 and said nucleotide sequence encodes apolypeptide having ubiquitin protease activity; (b) a nucleotidesequence complementary to the nucleotide sequence of (a).
 5. An isolatednucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence encoding a polypeptidehaving ubiquitin protease activity, wherein said nucleotide sequencehybridizes to the complement of the nucleotide sequence shown in SEQ IDNO:2 under stringent conditions, said stringent conditions comprisinghybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2 × SSC, 0.1% SDS at 65° C.; (b)a nucleotide sequence encoding a polypeptide having ubiquitin proteaseactivity, wherein said nucleotide sequence hybridizes to the cDNA insertcontained in ATCC Patent Deposit No. PTA-1652 under stringentconditions, said stringent conditions comprising hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2× SSC, 0.1% SDS at 65° C.; and (c) a nucleotidesequence complementary to the nucleotide sequence of (a) or (b).
 6. Anisolated nucleic acid molecule comprising a nucleotide sequence selectedfrom the group consisting of: (a) a nucleotide sequence encoding afragment of the amino acid sequence set forth in SEQ ID NO:1, whereinsaid fragment has ubiquitin protease activity and consists of at least15 contiguous amino acids of the amino acid sequence set forth in SEQ IDNO:1; (b) a nucleotide sequence encoding a fragment of the amino acidsequence encoded by the cDNA insert of the plasmid deposited with ATCCas Patent Deposit No. PTA-1652, wherein said fragment has ubiquitinprotease activity and consists of at least 15 contiguous amino acids ofthe amino acid sequence encoded by the cDNA insert of the plasmiddeposited with ATCC as Patent Deposit No. PTA-1652; (c) a nucleotidesequence encoding a fragment of the amino acid sequence set forth in SEQID NO:1, wherein said fragment has ubiquitin protease activity andconsists of at least 25 contiguous amino acids of the amino acidsequence set forth in SEQ ID NO:1; (d) a nucleotide sequence encoding afragment of the amino acid sequence encoded by the cDNA insert of theplasmid deposited with ATCC as Patent Deposit No. PTA-1652, wherein saidfragment has ubiquitin protease activity and consists of at least 25contiguous amino acids of the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with ATCC as Patent Deposit No.PTA-1652; (e) a nucleotide sequence encoding a fragment of the aminoacid sequence set forth in SEQ ID NO:1, wherein said fragment hasubiquitin protease activity and consists of at least 35 contiguous aminoacids of the amino acid sequence set forth in SEQ ID NO:1; and (f) anucleotide sequence encoding a fragment of the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with ATCC as PatentDeposit No. PTA-1652, wherein said fragment has ubiquitin proteaseactivity and consists of at least 35 contiguous amino acids of the aminoacid sequence encoded by the cDNA insert of the plasmid deposited withATCC as Patent Deposit No. PTA-1652; (g) a nucleotide sequence encodinga fragment of the amino acid sequence set forth in SEQ ID NO:1, whereinsaid fragment has ubiquitin protease activity and consists of at least45 contiguous amino acids of the amino acid sequence set forth in SEQ IDNO:1; and (h) a nucleotide sequence encoding a fragment of the aminoacid sequence encoded by the cDNA insert of the plasmid deposited withATCC as Patent Deposit No. PTA-1652, wherein said fragment has ubiquitinprotease activity and consists of at least 45 contiguous amino acids ofthe amino acid sequence encoded by the cDNA insert of the plasmiddeposited with ATCC as Patent Deposit No. PTA-1652.
 7. A method forproducing a polypeptide comprising an amino acid sequence selected fromthe group consisting of: (a) an amino acid sequence having at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO:1wherein said polypeptide has ubiquitin protease activity and saidsequence identity is calculated by the GAP algorithm using a Blossum 62scoring matrix with a gap weight of 12 and a length weight of 4; (b) anamino acid sequence having at least 80% sequence identity to the aminoacid sequence set forth in SEQ ID NO:1 wherein said polypeptide hasubiquitin protease activity and said sequence identity is calculated bythe GAP algorithm using a Blossum 62 scoring matrix with a gap weight of12 and a length weight of 4; (c) an amino acid sequence having at least90% sequence identity to the amino acid sequence set forth in SEQ IDNO:1 wherein said polypeptide has ubiquitin protease activity and saidsequence identity is calculated by the GAP algorithm using a Blossum 62scoring matrix with a gap weight of 12 and a length weight of 4; saidmethod comprising the steps of introducing a nucleic acid moleculeencoding the polypeptide into a host cell and culturing the host cellunder conditions in which the polypeptide is expressed from the nucleicacid molecule.
 8. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) the amino acidsequence set forth in SEQ ID NO:1; (b) the amino acid sequence encodedby the cDNA insert of the plasmid deposited with the ATCC as PatentDeposit Number PTA-1652; (c) the amino acid sequence of a sequencevariant of the amino acid sequence shown in SEQ ID NO:1, wherein saidsequence variant has ubiquitin protease activity and is encoded by anucleotide sequence having at least about 70% sequence identity to thenucleotide sequence set forth in SEQ ID NO:2; (d) the amino acidsequence of an sequence variant of the amino acid sequence encoded bythe cDNA insert of the plasmid deposited with the ATCC as Patent DepositNumber PTA-1652, wherein said sequence variant has ubiquitin proteaseactivity and is encoded by a nucleotide sequence having at least about70% sequence identity to the nucleotide sequence of the cDNA insert ofthe plasmid deposited with the ATCC as Patent Deposit Number PTA-1652;(e) the amino acid sequence of a sequence variant of the amino acidsequence shown in SEQ ID NO:1, wherein the sequence variant hasubiquitin protease activity and is encoded by a nucleic acid moleculethat hybridizes to the complement of the nucleotide sequence set forthin SEQ ID NO:2 under stringent conditions; (f) the amino acid sequenceof a sequence variant of the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with the ATCC as Patent Deposit NumberPTA-1652, wherein the sequence variant has ubiquitin protease activityand is encoded by a nucleic acid molecule that hybridizes understringent conditions the cDNA insert of the plasmid deposited with theATCC as Patent Deposit Number PTA-1652; (g) the amino acid sequence ofthe mature polypeptide from about amino acid 6 to the last amino acidset forth in SEQ ID NO:1; (h) the amino acid sequence of a fragment ofthe amino acid sequence set forth in SEQ ID NO:1, wherein said fragmenthas ubiquitin protease activity and consists of at least 15 contiguousamino acids of the amino acid sequence set forth in SEQ ID NO:1; and (i)the amino acid sequence of a fragment of the amino acid sequence encodedby the cDNA insert of the plasmid deposited with the ATCC as PatentDeposit Number PTA-1652, wherein the fragment has ubiquitin proteaseactivity and consists of at least 15 contiguous amino acids of the aminoacid sequence encoded by the cDNA insert of the plasmid deposited withthe ATCC as Patent Deposit Number PTA-1652.
 9. An isolated antibody thatselectively binds to a polypeptide of claim
 8. 10. A method fordetecting the presence of at least one polypeptides of claim 8 in asample, said method comprising contacting said sample with an agent thatspecifically allows detection of the presence of the polypeptide in thesample and then detecting the presence of the polypeptide.
 11. Themethod of claim 10, wherein said agent is an antibody.
 12. The method ofclaim 10, wherein said agent is ubiquitinated protein or polyubiquitin.13. A kit comprising reagents used for the method of claim 8, whereinthe reagents comprise an agent that specifically binds to saidpolypeptide.
 14. A method for identifying an agent that binds to any ofthe polypeptides in claim 8, said method comprising contacting thepolypeptide with an agent that binds to the polypeptide and assaying thecomplex formed with the agent bound to the polypeptide.
 15. A method foridentifying an agent that modulates the level or activity of any of thepolypeptides in claim 1 in a cell, the method comprising contacting theagent with a cell capable of expressing said polypeptide such that saidpolypeptide level or activity can be modulated in said cell by saidagent and measuring said polypeptide level or activity.
 16. A method foridentifying an agent that interacts with any of the polypeptides inclaim 8 in a cell, the method comprising contacting said agent with acell expressing a polypeptide of claim 8 under conditions such that saidpolypeptide can interact with said agent and measuring the interactionbetween said polypeptide and said agent.
 17. A method of screening acell to identify an agent that modulates the level or activity of any ofthe polypeptides in claim 8 in said cell, said method comprisingcontacting said agent with a cell capable of expressing said polypeptidesuch that said polypeptide level or activity can be modulated in saidcell by said agent and measuring said polypeptide level or activity. 18.A method of screening a cell to identify an agent that interacts withany of the polypeptides in claim 8, said method comprising contactingsaid agent with a cell capable of allowing an interaction between saidpolypeptide and said agent such that said polypeptide can interact withsaid agent and measuring the interaction.
 19. A method for modulatingthe activity of any of at least one polypeptide of claim 8, the methodcomprising contacting a polypeptide of claim 8 with an agent underconditions that allow the agent to modulate the activity of thepolypeptide.
 20. The method of claim 19 wherein said modulation is incells derived from tissues selected from the group consisting of breast,testes, brain, lung, kidney, liver, and colonic liver metastases. 21.The method of claim 20 wherein said cells are malignant breast, lung,and colonic liver metastatic cells.
 22. The method of claim 20 whereinsaid modulation is in a patient having breast cancer.
 23. A compositioncontaining any of the polypeptides in claim 8 in a pharmaceuticallyacceptable carrier.